Activin receptor-like kinase-1 compositions and methods of use

ABSTRACT

Disclosed herein are methods and compositions for treating disorders associated with angiogenesis or lymphangiogenesis using ALK-1 antagonists.

RELATED APPLICATION

This application claims priority to U.S. provisional application Ser.Nos. 60/986,876 filed on Nov. 9, 2007 and 61/098,557 filed on Sep. 19,2008, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention concerns methods and compositions relating to theuse of activin receptor-like kinase-1 (ALK-1) antagonists for thetreatment of disorders associated with angiogenesis and/orlymphangiogenesis.

BACKGROUND OF THE INVENTION

Development of a vascular supply is a fundamental requirement for manyphysiological and pathological processes. Actively growing tissues suchas embryos and tumors require adequate blood supply. They satisfy thisneed by producing pro-angiogenic factors, which promote new blood vesselformation via a process called angiogenesis. Vascular tube formation isa complex but orderly biological event involving all or many of thefollowing steps: a) Endothelial cells (ECs) proliferate from existingECs or differentiate from progenitor cells; b) ECs migrate and coalesceto form cord-like structures; c) vascular cords then undergotubulogenesis to form vessels with a central lumen; d) existing cords orvessels send out sprouts to form secondary vessels; e) primitivevascular plexus undergo further remodeling and reshaping; and f)peri-endothelial cells are recruited to encase the endothelial tubes,providing maintenance and modulatory functions to the vessels; suchcells including pericytes for small capillaries, smooth muscle cells forlarger vessels, and myocardial cells in the heart. Hanahan, D. Science277:48-50 (1997); Hogan, B. L. & Kolodziej, P. A. Nature ReviewsGenetics. 3:513-23 (2002); Lubarsky, B. & Krasnow, M. A. Cell. 112:19-28(2003).

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors andmetastasis, atherosclerosis, retrolental fibroplasia, hemangiomas,chronic inflammation, intraocular neovascular diseases such asproliferative retinopathies, e.g., diabetic retinopathy, age-relatedmacular degeneration (AMD), neovascular glaucoma, immune rejection oftransplanted corneal tissue and other tissues, rheumatoid arthritis, andpsoriasis. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992);Klagsbrun et al., Annu. Rev. Physiol. 53:217-239 (1991); and Garner A.,“Vascular diseases”, In: Pathobiology of Ocular Disease. A DynamicApproach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker,NY, 1994), pp 1625-1710.

In the case of tumor growth, angiogenesis appears to be crucial for thetransition from hyperplasia to neoplasia, and for providing nourishmentfor the growth and metastasis of the tumor. Folkman et al., Nature339:58 (1989). The neovascularization allows the tumor cells to acquirea growth advantage and proliferative autonomy compared to the normalcells. A tumor usually begins as a single aberrant cell which canproliferate only to a size of a few cubic millimeters due to thedistance from available capillary beds, and it can stay ‘dormant’without further growth and dissemination for a long period of time. Sometumor cells then switch to the angiogenic phenotype to activateendothelial cells, which proliferate and mature into new capillary bloodvessels. These newly formed blood vessels not only allow for continuedgrowth of the primary tumor, but also for the dissemination andrecolonization of metastatic tumor cells. Accordingly, a correlation hasbeen observed between density of microvessels in tumor sections andpatient survival in breast cancer as well as in several other tumors.Weidner et al., N. Engl. J. Med 324:1-6 (1991); Horak et al., Lancet340:1120-1124 (1992); Macchiarini et al., Lancet 340:145-146 (1992). Theprecise mechanisms that control the angiogenic switch is not wellunderstood, but it is believed that neovascularization of tumor massresults from the net balance of a multitude of angiogenesis stimulatorsand inhibitors (Folkman Nat Med 1(1):27-31 (1995)).

It is currently accepted that metastases are responsible for the vastmajority, estimated at 90%, of deaths from solid tumors (Gupta andMassague, Cell 127, 679-695 (2006)). The complex process of metastasisinvolves a series of distinct steps including detachment of tumor cellsfrom the primary tumor, intravasation of tumor cells into lymphatic orblood vessels, and extravasation and growth of tumor cells in secondarysites. Analysis of regional lymph nodes in many tumor types suggeststhat the lymphatic vasculature is an important route for thedissemination of human cancers. Furthermore, in almost all carcinomas,the presence of tumor cells in lymph nodes is the most important adverseprognostic factor. While it was previously thought that such metastasesexclusively involved passage of malignant cells along pre-existinglymphatic vessels near tumors, recent experimental studies andclinicopathological reports (reviewed in Achen et al., Br J Cancer 94(2006), 1355-1360 and Nathanson, Cancer 98, 413-423 (2003)) suggest thatlymphangiogenesis can be induced by solid tumors and can promote tumorspread. These and other recent studies suggest targeting lymphatics andlymphangiogenesis may be a useful therapeutic strategy to restrict thedevelopment of cancer metastasis, which would have a significant benefitfor many patients.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that agents thatmodulate the ALK-1 pathway are capable of impairing both angiogenesisand lymphangiogenesis. Treatment with an ALK-1 antagonist resulted inenhanced endothelial cell sprouting, disorganized pericytes, andimproper vascular development including tumor and lymphatic vasculature.Tumor models treated with an ALK-1 antagonist resulted in inhibition oftumor growth in several types of cancer. Accordingly, the inventionprovides methods, compositions, kits and articles of manufacture fordisrupting pericytes organization, inhibiting angiogenesis and/orlymphangiogenesis and for use in targeting pathological conditionsassociated with angiogenesis and/or lymphangiogenesis.

In one aspect the invention provides a method of inhibitinglymphangiogenesis comprising administering to a subject in need ofinhibition of lymphangiogenesis an effective amount of an ALK-1antagonist, whereby the lymphangiogensis is inhibited. In someembodiments the subject suffers from, e.g., a tumor, cancer, cellproliferative disorder, macular degeneration, inflammatory mediateddisease, rheumatoid arthritis, diabetic retinopathy or psoriasis. Insome embodiments the tumor, cancer or cell proliferative disorder iscarcinoma, lymphoma, blastoma, sarcoma, or leukemia.

In another aspect, the invention provides a method for treating apathological condition associated with lymphangiogenesis in a subjectcomprising administering to the subject an effective amount of an ALK-1antagonist, whereby the pathological condition associated withlymphangiogenesis is treated. The pathological condition associated withlymphangiogenesis may be, e.g., a tumor, cancer, cell proliferativedisorder, macular degeneration, inflammatory mediated disease,rheumatoid arthritis, diabetic retinopathy or psoriasis. In someembodiments the tumor, cancer or cell proliferative disorder iscarcinoma, lymphoma, blastoma, sarcoma, or leukemia.

The invention also provides use of an ALK-1 antagonist in thepreparation of a medicament for the therapeutic and/or prophylactictreatment of a disorder, such as a pathological condition associatedwith angiogenesis or lymphangiogenesis. Such conditions include, forexample, a tumor, cancer, cell proliferative disorder, maculardegeneration, inflammatory mediated disease, rheumatoid arthritis,diabetic retinopathy or psoriasis. Also provided is the use of an ALK-1antagonist in the preparation of a medicament for the therapeutic and/orprophylactic treatment of tumor metastasis.

In a further aspect the invention provides a method of inhibitingtumoral lymphangiogenesis in a subject comprising administering to thesubject an effective amount of an ALK-1 antagonist, whereby the tumorallymphangiogensis is inhibited. Also provided is a method of inhibitingor preventing tumor metastasis in a subject comprising administering tothe subject an effective amount of an ALK-1 antagonist, whereby thetumor metastasis is inhibited or prevented. In some embodiments thesubject has developed or is at risk for developing tumor metastasis. Thetumor metastasis may be, for example, in the lymphatic system or in adistant organ.

In another aspect the invention provides a method of disrupting pericyteorganization in a subject comprising administering to the subject aneffective amount of an ALK-1 antagonist, whereby the pericyteorganization is disrupted. In some embodiments the subject suffers from,e.g., a tumor, cancer, cell proliferative disorder, maculardegeneration, inflammatory mediated disease, rheumatoid arthritis,diabetic retinopathy or psoriasis. In some embodiments the tumor, canceror cell proliferative disorder is carcinoma, lymphoma, blastoma,sarcoma, or leukemia.

In yet another aspect, the invention provides a method of inhibitingtumor growth in a subject comprising administering to the subject aneffective amount of an ALK-1 antagonist, whereby the tumor growth isinhibited. The invention also provides a method of treating a tumor,cancer or cell proliferative disorder in a subject comprisingadministering to the subject an effective amount of an ALK-1 antagonist,whereby the tumor, cancer or cell proliferative disorder is treated. Insome embodiments, the tumor, cancer or cell proliferative disorder is,e.g., carcinoma, lymphoma, blastoma, sarcoma, or leukemia.

In some embodiments, the methods of the invention further compriseadministering to the subject an effective amount of an anti-angiogenesisagent. In some embodiments the anti-angiogenesis agent is an antagonistof vascular endothelial growth factor (VEGF), for example, an anti-VEGFantibody. In some embodiments the anti-VEGF antibody is bevacizumab. Insome embodiments, the methods of the invention further compriseadministering one or more chemotherapeutic agents.

The invention also provides a method of enhancing efficacy of ananti-angiogenesis agent in a subject having a pathological conditionassociated with angiogenesis, comprising administering to the subject aneffective amount of an ALK-1 antagonist in combination with theanti-angiogenesis agent, thereby enhancing said anti-angiogenesisagent's inhibitory activity. In some embodiments the pathologicalcondition associated with angiogenesis is a tumor, cancer or cellproliferative disorder.

In some embodiments the ALK-1 antagonist is an ALK-1 immunoadhesin. Insome embodiments the ALK-1 immunoadhesin comprises amino acid residues22-352 of SEQ ID NO: 2, residues 22-349 of SEQ ID NO: 4, residues 22-347of SEQ ID NO: 6, residues 22-350 of SEQ ID NO: 8 or residues 22-350 ofSEQ ID NO: 10. In other embodiments the ALK-1 antagonist is ananti-ALK-1 antibody or antigen-binding fragment thereof.

Also provided are ALK-1 antagonists for use in the prevention,inhibition or treatment of tumor metastasis or treatment of tumor,cancer or cell proliferative disorder. The ALK-1 antagonist may be, forexample, an ALK-1 immunoadhesin or an anti-ALK-1 antibody or antigenbinding fragment thereof. In some embodiments the ALK-1 immunoadhesincomprises amino acid residues 22-352 of SEQ ID NO: 2, resiudes 22-349 ofSEQ ID NO: 4, residues 22-347 of SEQ ID NO: 6, residues 22-350 of SEQ IDNO: 8 or residues 22-350 of SEQ ID NO: 10. The invention also provides acomposition for use in treating a tumor, cancer or cell proliferativedisorder comprising an effective amount of an ALK-1 antagonist and apharmaceutically acceptable carrier. In some embodiments the ALK-1antagonist is an ALK-1 immunoadhesin. In some embodiments the ALK-1immunoadhesin comprises amino acid residues 22-352 of SEQ ID NO: 2,resiudes 22-349 of SEQ ID NO: 4, residues 22-347 of SEQ ID NO: 6,residues 22-350 of SEQ ID NO: 8 or residues 22-350 of SEQ ID NO: 10. Insome embodiments the ALK-1 antagonist is an anti-ALK-1 antibody.

In one aspect the invention provides an article of manufacturecomprising a container and a composition contained within the container,wherein the composition comprises an ALK-1 antagonist. In another aspectthe invention provides a kit comprising an ALK-1 antagonist andinstructions for using the ALK-1 antagonist. Also provided is a methodfor preparing a composition comprising admixing a therapeuticallyeffective amount of an ALK-1 antagonist with a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are phase contract microscopy images taken on day 9 of HUVECsprouting assays in 3-D fibrin gels in the absence (FIG. 1A) or presence(FIG. 1B) of ALK1.Fc (5 μg/ml).

FIGS. 2A-2F are confocal images from rat retinas collected on P11 andstained with anti-CD31 and Lectin-FITC. Neonatal rats were injected i.p.with 10 mg/kg ALK1.Fc or PBS on postnatal day 4 (P4), P6 and P9.

FIGS. 3A-3F are confocal images from rat retinas collected on on P11 andstained with Cy3-conjugated anti-alpha smooth muscle actin (ASMA) andLectin-FITC. Neonatal rats were injected i.p. with 10 mg/kg ALK1.Fc orPBS on P4, P6 and P9.

FIGS. 4A-4F are confocal images from rat retinas collected on on P11 andstained with Cy3-conjugated anti-alpha smooth muscle actin (ASMA) andLectin-FITC. Neonatal rats were injected i.p. with 10 mg/kg ALK1.Fc orPBS on P4, P6 and P9.

FIGS. 5A-5B are graphs showing that ALK1.Fc (10 mg/kg) inhibits tumorgrowth in HM7 (FIG. 5A) and MV522 (FIG. 5B) xenografts. Mean tumorvolumes with SEs are presented.

FIGS. 6A-6B are graphs showing that ALK1.Fc (10 mg/kg) inhibits tumorgrowth in C6 (FIG. 6A) and Calu6 (FIG. 6B) xenografts. Mean tumorvolumes with SEs are presented.

FIG. 7 is a graph showing that ALK1.Fc has additive anti-tumor activitywhen combined with anti-VEGF in HM7 xenografts.

FIG. 8 is a graph showing that ALK1.Fc has additive anti-tumor activitywhen combined with anti-VEGF in LL2 xenografts.

FIGS. 9A-9D are confocal images showing tumor vascular density in LL2tumor model mice treated with PBS (FIG. 9A), ALK1.Fc (FIG. 9B),Anti-VEGF antibody (B20-4.1, 5 mg/kg) (FIG. 9C) or anti-VEGF antibodyand ALK1.Fc (FIG. 9D).

FIGS. 10A-10B are confocal images of intestinal villi from neonatal micetreated with PBS (FIG. 10A) or ALK1.Fc (FIG. 10B). The tissues werestained with anti-CD31 to show capillary blood vessels and anti-LYVE-1to show lymphatic capillaries.

FIGS. 11A-11B are confocal images of a single intestinal villi fromneonatal mice treated with PBS (FIG. 11A) or ALK1.Fc (FIG. 11B). Thetissues were stained with anti-CD31 to show capillary blood vessels andanti-LYVE-1 to show lymphatic capillaries.

FIGS. 12A-12F are confocal images of mouse tail skin from neonatal micetreated with PBS (FIG. 12A-C) or ALK1.Fc (FIG. 12D-F). The tissues werestained with anti-CD31 to show capillary blood vessels and anti-LYVE-1to show lymphatic capillaries.

FIGS. 13A-B are confocal images showing pericyte organization in TIB68tumor model mice treated with PBS (FIG. 13A) or ALK1.Fc (FIG. 13B)

FIG. 14 shows confocal images from mouse retinas collected on P8 andstained with anti-CD31. Mice were injected i.p. with PBS, ALK1.Fc,ALK1.Fc.2, ALK1.Fc.4 or ALK1.Fc.5 on P4 and P6.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994). Unless defined otherwise,technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), andMarch, Advanced Organic Chemistry Reactions, Mechanisms and Structure4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DEFINITIONS

The term “ALK-1” (interchangeably termed “activin receptor-likekinase-1”), as used herein, refers, unless specifically or contextuallyindicated otherwise, to any native or variant (whether native orsynthetic) ALK-1 polypeptide. The term “native sequence” specificallyencompasses naturally occurring truncated or secreted forms (e.g., anextracellular domain sequence), naturally occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants.The term “wild type ALK-1” generally refers to a polypeptide comprisingthe amino acid sequence of a naturally occurring ALK-1 protein. The term“wild type ALK-1 sequence” generally refers to an amino acid sequencefound in a naturally occurring ALK-1.

A “chimeric ALK-1” molecule is a polypeptide comprising full-lengthALK-1 or one or more domains thereof fused or bonded to heterologouspolypeptide. The chimeric ALK-1 molecule will generally share at leastone biological property in common with naturally occurring ALK-1. Anexample of a chimeric ALK-1 molecule is one that is epitope tagged forpurification purposes. Another chimeric ALK-1 molecule is an ALK-1immunoadhesin.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The term “ALK-1 immunoadhesin” is used interchangeably with the term“ALK-1-immunoglobulin chimera”, and refers to a chimeric molecule thatcombines at least a portion of an ALK-1 molecule (native or variant)with an immunoglobulin sequence. In some instances the ALK-1immunoadhesin comprises the extracellular domain (ECD) of ALK-1 or aportion thereof sufficient to bind to ALK-1 ligand. In some embodimentsthe N-terminus of the ALK-1 portion of the ALK-1 immunoadhesin may beginat any amino acid residue from Asp 22 to Ser 27 (inclusive) of SEQ IDNO:2 and the C-terminus of the ALK-1 portion of the ALK-1 immunoadhesinmay end at any amino acid residue from Ser 110 to Glu 118 of SEQ DI NO:2 (inclusive). In some embodiments, the ALK-1 immunoadhesin comprisesamino acid residues 1-118 of SEQ ID NO:2. In some embodiments the ALK-1immunoadhesin comprises amino acid residues 22-118 of SEQ ID NO:2,residues 23-118 of SEQ ID NO: 2, residues 24-118 of SEQ ID NO: 2,residues 25-118 of SEQ ID NO: 2, residues 26-118 of SEQ ID NO: 2, orresidues 27-118 of SEQ ID NO: 2. In some embodiments the unprocessedALK-1 immunoadhesin is ALK1.Fc (SEQ ID NO: 2), ALK1.Fc.2 (SEQ ID NO: 4),ALK1.Fc.3 (SEQ ID NO: 6), ALK1.Fc.4 (SEQ ID NO: 8) or ALK1.Fc.5 (SEQ IDNO: 10). In some embodiments the ALK-1 immunoadhesin comprises aminoacid residues 22-352 of SEQ ID NO: 2, residues 22-349 of SEQ ID NO: 4,residues 22-347 of SEQ ID NO: 6, residues 22-350 of SEQ ID NO: 8 orresidues 22-350 of SEQ ID NO: 10. The immunoglobulin sequencepreferably, but not necessarily, is an immunoglobulin constant domain(Fc region). Immunoadhesins can possess many of the valuable chemicaland biological properties of human antibodies. Since immunoadhesins canbe constructed from a human protein sequence with a desired specificitylinked to an appropriate human immunoglobulin hinge and constant domain(Fc) sequence, the binding specificity of interest can be achieved usingentirely human components. Such immunoadhesins are minimally immunogenicto the patient, and are safe for chronic or repeated use. In someembodiments, the Fc region is a native sequence Fc region. In someembodiments, the Fc region is a variant Fc region. In some embodiments,the Fc region is a functional Fc region. The ALK-1 portion and theimmunoglobulin sequence portion of the ALK-1 immunoadhesin may be linkedby a minimal linker.

Examples of homomultimeric immunoadhesins which have been described fortherapeutic use include the CD4-IgG immunoadhesin for blocking thebinding of HIV to cell-surface CD4. Data obtained from Phase I clinicaltrials, in which CD4-IgG was administered to pregnant women just beforedelivery, suggests that this immunoadhesin may be useful in theprevention of maternal-fetal transfer of HIV (Ashkenazi et al., Intern.Rev. Immunol. 10:219-227 (1993)). An immunoadhesin which binds tumornecrosis factor (TNF) has also been developed. TNF is a proinflammatorycytokine which has been shown to be a major mediator of septic shock.Based on a mouse model of septic shock, a TNF receptor immunoadhesin hasshown promise as a candidate for clinical use in treating septic shock(Ashkenazi, A. et al. PNAS USA 88:10535-10539 (1991)). ENBREL®(etanercept), an immunoadhesin comprising a TNF receptor sequence fusedto an IgG Fc region, was approved by the U.S. Food and DrugAdministration (FDA), on Nov. 2, 1998, for the treatment of rheumatoidarthritis. The new expanded use of ENBREL® in the treatment ofrheumatoid arthritis was approved by FDA on Jun. 6, 2000. For recentinformation on TNF blockers, including ENBREL®, see Lovell et al., N.Engl. J. Med. 342:763-169 (2000), and accompanying editorial on p810-811; and Weinblatt et al., N. Engl. J. Med. 340:253-259 (1999);reviewed in Maini and Taylor, Annu. Rev. Med. 51:207-229 (2000).

If the two arms of the immunoadhesin structure have differentspecificities, the immunoadhesin is called a “bispecific immunoadhesin”by analogy to bispecific antibodies. Dietsch et al., J. Immunol. Methods162:123 (1993) describe such a bispecific immunoadhesin combining theextracellular domains of the adhesion molecules, E-selectin andP-selectin, each of which selectins is expressed in a different celltype in nature. Binding studies indicated that the bispecificimmunoglobulin fusion protein so formed had an enhanced ability to bindto a myeloid cell line compared to the monospecific immunoadhesins fromwhich it was derived.

The term “heteroadhesin” is used interchangeably with the expression“chimeric heteromultimer adhesin” and refers to a complex of chimericmolecules (amino acid sequences) in which each chimeric moleculecombines a biologically active portion, such as the extracellular domainof each of the heteromultimeric receptor monomers, with amultimerization domain. The “multimerization domain” promotes stableinteraction of the chimeric molecules within the heteromultimer complex.The multimerization domains may interact via an immunoglobulin sequence,leucine zipper, a hydrophobic region, a hydrophilic region, or a freethiol that forms an intermolecular disulfide bond between the chimericmolecules of the chimeric heteromultimer. The multimerization domain maycomprise an immunoglobulin constant region. In addition amultimerization region may be engineered such that steric interactionsnot only promote stable interaction, but further promote the formationof heterodimers over homodimers from a mixture of monomers.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the protuberances are optionally created onthe interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). Theimmunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG₁, IgG₂, IgG₃or IgG₄ subtypes, IgA, IgE, IgD or IgM, but preferably IgG₁ or IgG₃.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

Unless indicated otherwise, herein the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991), expresslyincorporated herein by reference. The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In one embodiment, an antibody fragment comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, an antibody fragment, for example onethat comprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcRn binding, antibody half life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For e.g., such an antibodyfragment may comprise on antigen binding arm linked to an Fc sequencecapable of conferring in vivo stability to the fragment.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH(H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

The term “monoclonal antibody” as used herein refers to an antibody froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical and/orbind the same epitope(s), except for possible variants that may ariseduring production of the monoclonal antibody, such variants generallybeing present in minor amounts. Such monoclonal antibody typicallyincludes an antibody comprising a polypeptide sequence that binds atarget, wherein the target-binding polypeptide sequence was obtained bya process that includes the selection of a single target bindingpolypeptide sequence from a plurality of polypeptide sequences. Forexample, the selection process can be the selection of a unique clonefrom a plurality of clones, such as a pool of hybridoma clones, phageclones or recombinant DNA clones. It should be understood that theselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity, themonoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler et al.,Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier,N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567), phage display technologies (see, e.g., Clackson et al.,Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597(1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al.,J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci.USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods284(1-2):119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669(all of GenPharm); 5,545,807; WO 1997/17852; U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al.,Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859(1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., NatureBiotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intem. Rev. Immunol., 13: 65-93(1995).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation. Antibody-dependent cell-mediated cytotoxicity” or “ADCC”refers to a form of cytotoxicity in which secreted Ig bound onto Fcreceptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer(NK) cells, neutrophils, and macrophages) enable these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell with cytotoxins. The antibodies“arm” the cytotoxic cells and are absolutely required for such killing.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRII, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of amolecule of interest, an in vitro ADCC assay, such as that described inU.S. Pat. No. 5,500,362 or 5,821,337 or Presta U.S. Pat. No. 6,737,056may be performed. Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins.

WO00/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. The content of that patent publication isspecifically incorporated herein by reference. See, also, Shields et al.J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin, which comprises an Fc region. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during purification of thepolypeptide or by recombinant engineering the nucleic acid encoding thepolypeptide. Accordingly, a composition comprising a polypeptide havingan Fc region according to this invention can comprise polypeptides withK447, with all K447 removed, or a mixture of polypeptides with andwithout the K447 residue.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors, carcinoma, blastoma, and sarcoma.Conditions associated with abnormal or excessive lymphangiogenesisinclude, without limitation, tumors, cancer, cell proliferativedisorders, macular degeneration, inflammatory mediated disease,rheumatoid arthritis, diabetic retinopathy and psoriasis.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and varioustypes of head and neck cancer. Dysregulation of angiogenesis can lead tomany disorders that can be treated by compositions and methods of theinvention. These disorders include both non-neoplastic and neoplasticconditions. Neoplastic disorders include but are not limited thosedescribed above.

Non-neoplastic disorders include but are not limited to undesired oraberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis,psoriatic plaques, sarcoidosis, atherosclerosis, atheroscleroticplaques, diabetic and other proliferative retinopathies includingretinopathy of prematurity, retrolental fibroplasia, neovascularglaucoma, age-related macular degeneration, diabetic macular edema,corneal neovascularization, corneal graft neovascularization, cornealgraft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, chronicinflammation, lung inflammation, acute lung injury/ARDS, sepsis, primarypulmonary hypertension, malignant pulmonary effusions, cerebral edema(e.g., associated with acute stroke/closed head injury/trauma), synovialinflammation, pannus formation in RA, myositis ossificans, hypertropicbone formation, osteoarthritis (OA), refractory ascites, polycysticovarian disease, endometriosis, 3rd spacing of fluid diseases(pancreatitis, compartment syndrome, burns, bowel disease), uterinefibroids, premature labor, chronic inflammation such as IBD (Crohn'sdisease and ulcerative colitis), renal allograft rejection, inflammatorybowel disease, nephrotic syndrome, undesired or aberrant tissue massgrowth (non-cancer), hemophilic joints, hypertrophic scars, inhibitionof hair growth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies areused to delay development of a disease or disorder.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows and sheep), sport animals, pets (such as cats, dogs and horses),primates, mice and rats.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the substance/molecule, agonist orantagonist to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the substance/molecule, agonist or antagonist areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSKR polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINEL®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRONR®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells (such as a cell expressing ALK-1) in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxanes, and topoisomerase IIinhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, andbleomycin. Those agents that arrest G1 also spill over into S-phasearrest, for example, DNA alkylating agents such as tamoxifen,prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate,5-fluorouracil, and ara-C. Further information can be found in TheMolecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” byMurakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. Thetaxanes (paclitaxel and docetaxel) are anticancer drugs both derivedfrom the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derivedfrom the European yew, is a semisynthetic analogue of paclitaxel(TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote theassembly of microtubules from tubulin dimers and stabilize microtubulesby preventing depolymerization, which results in the inhibition ofmitosis in cells.

The “pathology” of a disease includes all phenomena that compromise thewell-being of the patient. For cancer, this includes, withoutlimitation, abnormal or uncontrollable cell growth, metastasis,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels, suppressionor aggravation of inflammatory or immunological response, etc.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a ALK-1 polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

The terms “VEGF” and “VEGF-A” are used interchangeably to refer to the165-amino acid vascular endothelial cell growth factor and related 121-,145-, 183-, 189-, and 206-amino acid vascular endothelial cell growthfactors, as described by Leung et al. Science, 246:1306 (1989), Houck etal. Mol. Endocrin., 5:1806 (1991), and, Robinson & Stringer, Journal ofCell Science, 144(5):853-865 (2001), together with the naturallyoccurring allelic and processed forms thereof.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including its binding to one or more VEGF receptors. VEGFantagonists include anti-VEGF antibodies and antigen-binding fragmentsthereof, receptor molecules and derivatives which bind specifically toVEGF thereby sequestering its binding to one or more receptors,anti-VEGF receptor antibodies and VEGF receptor antagonists such assmall molecule inhibitors of the VEGFR tyrosine kinases, and fusionsproteins, e.g., VEGF-Trap (Regeneron), VEGF 121-gelonin (Peregrine).VEGF antagonists also include antagonist variants of VEGF, antisensemolecules directed to VEGF, RNA aptamers, and ribozymes against VEGF orVEGF receptors.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. The anti-VEGF antibody can be usedas a therapeutic agent in targeting and interfering with diseases orconditions wherein the VEGF activity is involved. See, e.g., U.S. Pat.Nos. 6,582,959, 6,703,020; WO98/45332; WO 96/30046; WO94/10202,WO2005/044853; EP 0666868B1; US Patent Applications 20030206899,20030190317, 20030203409, 20050112126, 20050186208, and 20050112126;Popkov et al., Journal of Immunological Methods 288:149-164 (2004); andWO2005012359. An anti-VEGF antibody will usually not bind to other VEGFhomologues such as VEGF-B or VEGF-C, nor other growth factors such asP1GF, PDGF or bFGF. The anti-VEGF antibody “Bevacizumab (BV)”, alsoknown as “rhuMAb VEGF” or “Avastin®”, is a recombinant humanizedanti-VEGF monoclonal antibody generated according to Presta et al.Cancer Res. 57:4593-4599 (1997). It comprises mutated human IgG1framework regions and antigen-binding complementarity-determiningregions from the murine anti-hVEGF monoclonal antibody A.4.6.1 thatblocks binding of human VEGF to its receptors. Approximately 93% of theamino acid sequence of Bevacizumab, including most of the frameworkregions, is derived from human IgG1, and about 7% of the sequence isderived from the murine antibody A4.6.1. Bevacizumab has a molecularmass of about 149,000 daltons and is glycosylated. Bevacizumab and otherhumanized anti-VEGF antibodies, including the anti-VEGF antibodyfragment “ranibizumab”, also known as “Lucentis®”, are further describedin U.S. Pat. No. 6,884,879 issued Feb. 26, 2005.

The term “biological activity” and “biologically active” with regard toan ALK-1 polypeptide refer to physical/chemical properties andbiological functions associated with ALK-1. In some embodiments, ALK-1“biological activity” includes one or more of: binding to an ALK-1ligand, e.g., BMP9 and/or BMP10 or activating ALK-1 downstream molecularsignaling, e.g., phosphorylation of Smad 1, Smad5 and/or Smad8.

A “ALK-1 antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with theactivities of a ALK-1 including, for example, reduction or blocking ofALK-1 receptor activation, reduction or blocking of ALK-1 downstreammolecular signaling, e.g., phosphorylation of Smad1, Smad5 and/or Smad8,disruption or blocking of ALK-1 ligand (e.g., BMP9 or BMP 10) binding toALK-1. ALK-1 antagonists include antibodies and antigen-bindingfragments thereof, proteins, peptides, glycoproteins, glycopeptides,glycolipids, polysaccharides, oligosaccharides, nucleic acids,bioorganic molecules, peptidomimetics, pharmacological agents and theirmetabolites, transcriptional and translation control sequences, and thelike. Antagonists also include small molecule inhibitors of a protein,and fusions proteins (including immunoadhesins), receptor molecules andderivatives which bind specifically to protein thereby sequestering itsbinding to its target, antagonist variants of the protein, siRNAmolecules directed to a protein, antisense molecules directed to aprotein, RNA aptamers, and ribozymes against a protein. In someembodiments, the ALK-1 antagonist is a molecule which binds to ALK-1 andneutralizes, blocks, inhibits, abrogates, reduces or interferes with abiological activity of ALK-1.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent”. Examples of therapeutic agents (anti-cancer agents,also termed “anti-neoplastic agent” herein) include, but are limited to,e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxicagents, agents used in radiation therapy, anti-angiogenesis agents,apoptotic agents, anti-tubulin agents, toxins, and other-agents to treatcancer, e.g., anti-VEGF neutralizing antibody, VEGF antagonist,anti-HER-2, anti-CD20, an epidermal growth factor receptor (EGFR)antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor,erlotinib, a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe ErbB2, ErbB3, ErbB4, or VEGF receptor(s), inhibitors for receptortyrosine kinases for platet-derived growth factor (PDGF) and/or stemcell factor (SCF) (e.g., imatinib mesylate (Gleevec R Novartis)),TRAIL/Apo2L, and other bioactive and organic chemical agents, etc.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promotes angiogenesis, endothelialcell growth, stability of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, P1GF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, ANGPTL3,ALK-1, etc. It would also include factors that accelerate wound healing,such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF,epidermal growth factor (EGF), CTGF and members of its family, and TGF-αand TGF-β. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol.,53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003);Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini etal., Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listing angiogenicfactors); and, Sato Int. J. Clin. Oncol., 8:200-206 (2003).

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide (including, e.g., aninhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, arecombinant protein, an antibody, or conjugates or fusion proteinsthereof, that inhibits angiogenesis, vasculogenesis, or undesirablevascular permeability, either directly or indirectly. For example, ananti-angiogenesis agent is an antibody or other antagonist to anangiogenic agent as defined above, e.g., antibodies to VEGF, antibodiesto VEGF receptors, small molecules that block VEGF receptor signaling(e.g., PTK787/ZK2284, SU6668, SUTENT®/SU11248 (sunitinib malate),AMG706, or those described in, e.g., international patent application WO2004/113304). Anti-angiogensis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar,Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenictherapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003)(e.g., Table 2 listing antiangiogenic factors); and, Sato Int. J. Clin.Oncol., 8:200-206 (2003) (e.g., Table 1 lists Anti-angiogenesis agentsused in clinical trials).

Methods and Compositions of the Invention

The present invention is based in part on the discovery that agents thatmodulate the ALK-1 pathway (e.g., an ALK-1 immunoadhesin or anti-ALK-1antibody) are capable of affecting both angiogenesis as well alymphangiogenesis. In one aspect the invention provides a method ofpreventing differentiation of endothelial cells into a more mature stageof differentiation comprising contacting the endothelial cells with anALK-1 antagonist. The endothelial cells may be any type of endothelialcells, e.g., blood or lymphatic endothelial cells. In another aspect theinvention provides a method of disrupting pericyte organizationcomprising contacting the pericytes with an ALK-1 antagonist. Inaddition, such agents were found to be able to inhibit tumor growth bothas a single agent and in combination with a VEGF antagonist. AccordinglyALK-1 antagonists are useful for pathological conditions and disordersassociated with angiogenesis as well as lymphangiogenesis.

It is contemplated that ALK-1 antagonists can be used to treat orprevent various pathological conditions or disorders. The inventionencompasses a method of inhibiting lymphangiogenesis using an effectiveamount of an ALK-1 antagonist such as, without limitation, an ALK-1immunoadhesin or anti-ALK-1 antibody, to inhibit activation of the ALK-1receptor pathway. In another aspect the invention provides a method ofinhibiting lymphangiogenesis comprising administering an effectiveamount of an ALK-1 antagonist to a subject in need of such treatment.

Examples of pathological conditions or disorders associated withabnormal lymphangiogenesis include, without limitation, tumor, cancer,tumor or cancer metastasis, cell proliferative disorder, maculardegeneration, inflammatory mediated disease, rheumatoid arthritis,diabetic retinopathy and psoriasis. In one aspect the invention providesa method of inhibiting or preventing tumoral lymphangiogenesis in asubject comprising administering to the subject an effective amount ofan ALK-1 antagonist. Also provided is a method of inhibiting orpreventing tumor metastasis in a subject comprising administering to thesubject an effective amount of an ALK-1 antagonist. In some embodiments,the subject may have developed or be at risk for developing tumormetastasis. Such metastasis may be in the lymphatic system or in adistant organ.

The invention provides a method of disrupting pericyte organization in asubject comprising administering to the subject an effective amount ofan ALK-1 antagonist. In some embodiments the subject suffers from tumor,cancer, tumor or cancer metastasis, cell proliferative disorder, maculardegeneration, inflammatory mediated disease, rheumatoid arthritis,diabetic retinopathy and psoriasis. In some embodiments the disruptionof pericyte organization occurs in the tumor vessels of a subjectafflicted with neoplastic disease such as a tumor or cancer.

The invention contemplates a method of treating tumor, cancer, cellproliferative disorder and/or neoplastic disorder in a subjectcomprising administering to the subject an effective amount of an ALK-1antagonist. In one aspect the invention provides a method of inhibitingtumor growth in a subject comprising administering an effective amountof an ALK-1 antagonist. Examples of neoplastic disorders to be treatedwith an ALK-1 antagonist include, but are not limited to, thosedescribed herein under the terms “cancer” and “cancerous.”

Non-neoplastic conditions that are amenable to treatment with ALK-1antagonists useful in the invention, include but are not limited to,e.g., undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis(RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis,atherosclerotic plaques, edema from myocardial infarction, diabetic andother proliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, diabetic macular edema, corneal neovascularization,corneal graft neovascularization, corneal graft rejection,retinal/choroidal neovascularization, neovascularization of the angle(rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis (OA), refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), obesity, adipose tissue mass growth, hemophilic joints,hypertrophic scars, inhibition of hair growth, Osler-Weber syndrome,pyogenic granuloma retrolental fibroplasias, scleroderma, trachoma,vascular adhesions, synovitis, dermatitis, preeclampsia, ascites,pericardial effusion (such as that associated with pericarditis), andpleural effusion. Further examples of disorders include an epithelial orcardiac disorder.

Combination Therapies

As indicated above, the invention provides combined therapies in which aALK-1 antagonist (such as an ALK-1 immunoadhesin or anti-ALK-1 antibody)is administered with another therapy. For example, ALK-1 antagonists areused in combinations with anti-cancer agent or an anti-angiogenesisagent to treat various neoplastic or non-neoplastic conditions. Inanother example, ALK-1 antagonists are used in combination withanti-lymphangiogenic agents (e.g., a VEGFC antagonist such as ananti-VEGFC antibody). In one embodiment, the neoplastic ornon-neoplastic condition is characterized by pathological disorderassociated with aberrant or undesired angiogenesis or lymphangiogenesis.The ALK-1 antagonist can be administered serially or in combination withanother agent that is effective for those purposes, either in the samecomposition or as separate compositions. Alternatively, or additionally,multiple inhibitors of ALK-1 can be administered.

The administration of the ALK-1 antagonist and the other therapeuticagent (e.g., anti-cancer agent, anti-angiogenesis agent) can be carriedout simultaneously, e.g., as a single composition or as two or moredistinct compositions using the same or different administration routes.Alternatively, or additionally, the administration can be donesequentially, in any order. Alternatively, or additionally, the stepscan be performed as a combination of both sequentially andsimultaneously, in any order.

In certain embodiments, intervals ranging from minutes to days, to weeksto months, can be present between the administrations of the two or morecompositions. For example, the anti-cancer agent may be administeredfirst, followed by the ALK-1 antagonist. However, simultaneousadministration or administration of the ALK-1 antagonist first is alsocontemplated. Accordingly, in one aspect, the invention provides methodscomprising administration of an ALK-1 antagonist (such as an ALK-1immunoadhesin or anti-ALK-1 antibody), followed by administration of ananti-angiogenesis agent (such as a VEGF antagonist, e.g., an anti-VEGFantibody). In one embodiment, the anti-angiogenesis agent is ananti-VEGF neutralizing antibody or fragment (e.g., humanized A4.6.1,AVASTIN® (Genentech, South San Francisco, Calif.), Y0317, M4, G6, B20,2C3, etc.). See, e.g., U.S. Pat. Nos. 6,582,959, 6,884,879, 6,703,020;WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1; US PatentApplications 20030206899, 20030190317, 20030203409, and 20050112126;Popkov et al., Journal of Immunological Methods 288:149-164 (2004); and,WO2005012359.

The effective amounts of therapeutic agents administered in combinationwith an ALK-1 antagonist will be at the physician's or veterinarian'sdiscretion. Dosage administration and adjustment is done to achievemaximal management of the conditions to be treated. The dose willadditionally depend on such factors as the type of therapeutic agent tobe used and the specific patient being treated. Suitable dosages for theanti-cancer agent are those presently used and can be lowered due to thecombined action (synergy) of the anti-cancer agent and the ALK-1antagonist. In certain embodiments, the combination of the inhibitorspotentiates the efficacy of a single inhibitor. The term “potentiate”refers to an improvement in the efficacy of a therapeutic agent at itscommon or approved dose. See also the section entitled PharmaceuticalCompositions herein.

Typically, the ALK-1 antagonists and anti-cancer agents are suitable forthe same or similar diseases to block or reduce a pathological disordersuch as a tumor, a cancer or a cell proliferative disorder. In oneembodiment the anti-cancer agent is an anti-angiogenesis agent.

Antiangiogenic therapy in relationship to cancer is a cancer treatmentstrategy aimed at inhibiting the development of tumor blood vesselsrequired for providing nutrients to support tumor growth. Becauseangiogenesis is involved in both primary tumor growth and metastasis,the antiangiogenic treatment provided by the invention is capable ofinhibiting the neoplastic growth of tumor at the primary site as well aspreventing metastasis of tumors at the secondary sites, thereforeallowing attack of the tumors by other therapeutics.

Many anti-angiogenesis agents have been identified and are known in thearts, including those listed herein, e.g., listed under the Definitionssection, and by, e.g., Carmeliet and Jain, Nature 407:249-257 (2000);Ferrara et al., Nature Reviews:Drug Discovery, 3:391-400 (2004); andSato Int. J. Clin. Oncol., 8:200-206 (2003). See also, US PatentApplication US20030055006. In one embodiment, an ALK-1 antagonist isused in combination with an anti-VEGF neutralizing antibody (orfragment) and/or another VEGF antagonist or a VEGF receptor antagonistincluding, but not limited to, for example, soluble VEGF receptor (e.g.,VEGFR-1, VEGFR-2, VEGFR-3, neuropillins (e.g., NRP1, NRP2)) fragments,aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFRantibodies, low molecule weight inhibitors of VEGFR tyrosine kinases(RTK), antisense strategies for VEGF, ribozymes against VEGF or VEGFreceptors, antagonist variants of VEGF; and any combinations thereof.Alternatively, or additionally, two or more angiogenesis inhibitors mayoptionally be co-administered to the patient in addition to VEGFantagonist and other agent. In certain embodiment, one or moreadditional therapeutic agents, e.g., anti-cancer agents, can beadministered in combination with ALK-1 antagonist, the VEGF antagonist,and an anti-angiogenesis agent.

In certain aspects of the invention, other therapeutic agents useful forcombination tumor therapy with an ALK-1 antagonist include other cancertherapies, (e.g., surgery, radiological treatments (e.g., involvingirradiation or administration of radioactive substances), chemotherapy,treatment with anti-cancer agents listed herein and known in the art, orcombinations thereof). Alternatively, or additionally, two or moreantibodies binding the same or two or more different antigens disclosedherein can be co-administered to the patient. Sometimes, it may bebeneficial to also administer one or more cytokines to the patient.

Chemotherapeutic Agents

In one aspect, the invention provides a method of treating a disorder(such as a tumor, a cancer, or a cell proliferative disorder) byadministering effective amounts of an ALK-1 antagonist (and/or an angiogenesis inhibitor(s)) and one or more chemotherapeutic agents. A varietyof chemotherapeutic agents may be used in the combined treatment methodsof the invention. An exemplary and non-limiting list of chemotherapeuticagents contemplated is provided herein under “Definitions.” Theadministration of the ALK-1 antagonist and the chemotherapeutic agentcan be done simultaneously, e.g., as a single composition or as two ormore distinct compositions, using the same or different administrationroutes. Alternatively, or additionally, the administration can be donesequentially, in any order. Alternatively, or additionally, the stepscan be performed as a combination of both sequentially andsimultaneously, in any order. In certain embodiments, intervals rangingfrom minutes to days, to weeks to months, can be present between theadministrations of the two or more compositions. For example, thechemotherapeutic agent may be administered first, followed by the ALK-1antagonist. However, simultaneous administration or administration ofthe ALK-1 antagonist prior to administration of the chemotherapeuticagent is also contemplated. Accordingly, in one aspect, the inventionprovides methods comprising administration of a ALK-1 antagonist (suchas an ALK-1 immunoadhesin or anti-ALK-1 antibody), followed byadministration of a chemotherapeutic agent.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. Variation in dosage will likely occur depending onthe condition being treated. The physician administering treatment willbe able to determine the appropriate dose for the individual subject.

ALK-1

ALK-1 is an endothelial-specific type I receptor of the TGFβ receptorfamily. The ALK-1 gene encodes a 503 amino acid polypeptide and includesa hydrophilic cysteine-rich ligand-binding domain, a single hydrophobictransmembrane region and a C-terminal intracellular portion thatconsists mainly of serine/threonine kinase domains. The accession numberof human ALK-1 is CAA80255 and the accession number of mouse ALK-1 isCAA83484.

Mutations in ALK-1 have been associated with hereditary hemorrhagictelangiectasia type 2. For many years ALK-1 was an orphan receptor.Although TGFβ1 and TGFβ3 were previously speculated to be ALK-1 ligands,recently BMP9 and BMP10 have been identified as the physiologicalligands of ALK-1 (David, L. et al., Blood 109:1953-1961 (2007)).Activation of ALK-1 has been shown to induce phosphrylation of Smad1,Smad5 and Smad8.

ALK-1 Antagonists

An exemplary and non-limiting list of ALK-1 antagonists (such as ananti-ALK-1 antibody and an ALK-1 immunoadhesin) contemplated is providedherein under “Definitions.”

The ALK-1 antagonists useful in the present invention can becharacterized for their physical/chemical properties and biologicalfunctions by various assays known in the art. In some embodiments, ALK-1antagonists are characterized for any one or more of: binding to ALK-1,binding to one or more ALK-1 ligands, eg., BMP9 or BMP10, reduction orblocking of ALK-1 receptor activation, reduction or blocking of ALK-1downstream molecular signaling (e.g., phospharylation of Smad 1, Smad 5and/or Smad 8), disruption or blocking of BMP9 or BMP 10 binding toALK-1, inhibition of angiogenesis, inhibition of lymphangiogenesis,treatment and/or prevention of a tumor, cell proliferative disorder or acancer; treatment or prevention of a disorder associated with ALK-1expression. Methods for characterizing ALK-1 antagonists are known inthe art, and some are described and exemplified herein.

ALK-1 Immunoadhesins

Immunoadhesins, including their structure and preparation, aredescribed, e.g. in WO 91/08298; and in U.S. Pat. Nos. 5,428,130 and5,116,964, the disclosures of which are hereby expressly incorporated byreference.

Production of an Immunoadhesin or Chimeric Heteromultimer Adhesin

The description below relates primarily to production of immunoadhesinby culturing cells transformed or transfected with a vector containingimmunoadhesin nucleic acid. It is, of course, contemplated thatalternative methods, which are well known in the art, may be employed toprepare immunoadhesins. For instance, the immunoadhesin sequence, orportions thereof, may be produced by direct peptide synthesis usingsolid-phase techniques (see, e.g., Stewart et al., Solid-Phase PeptideSynthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield,J. Am. Chem. Soc., 85:2149-2154 (1963)). In vitro protein synthesis maybe performed using manual techniques or by automation. Automatedsynthesis may be accomplished, for instance, using an Applied BiosystemsPeptide Synthesizer (Foster City, Calif.) using manufacturer'sinstructions. Various portions of the immunoadhesin may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length immunoadhesin.

Nucleic acid encoding a native sequence ALK-1 receptor can, for example,be isolated from cells known to express the ALK-1 receptor. Theaccession number for human ALK-1 cDNA is Z22533 and the number for mouseALK-1 cDNA is Z31664.1.

DNA encoding immunoglobulin light or heavy chain constant regions isknown or readily available from cDNA libraries or is synthesized. Seefor example, Adams et al., Biochemistry 19:2711-2719 (1980); Gough etal., Biochemistry 19:2702-2710 (1980); Dolby et al; P.N.A.S. USA,77:6027-6031 (1980); Rice et al P.N.A.S USA 79:7862-7865 (1982); Falkneret al; Nature 298:286-288 (1982); and Morrison et al; Ann. Rev. Immunol.2:239-256 (1984).

An immunoadhesin or a chimeric heteroadhesin of the invention ispreferably produced by expression in a host cell and isolated therefrom.A host cell is generally transformed with the nucleic acid of theinvention. Preferably the nucleic acid is incorporated into anexpression vector. Suitable host cells for cloning or expressing thevectors herein include prokaryotic host cells (such as E. coli, strainsof Bacillus, Pseudomonas and other bacteria), yeast and other eukaryoticmicrobes, and higher eukaryote cells (such as Chinese hamster ovary(CHO) cells and other mammalian cells). The cells may also be present inlive animals (for example, in cows, goats or sheep). Insect cells mayalso be used. Cloning and expression methodologies are well known in theart.

To obtain expression of an immunoadhesin such as the ALK1.Fc molecule(described in detail in Example 1), one or more expression vector(s)is/are introduced into host cells by transformation or transfection andthe resulting recombinant host cells are cultured in conventionalnutrient media, modified as appropriate for inducing promoters,selecting recombinant cells, or amplifying the ALK1.Fc DNA. In general,principles, protocols, and practical techniques for maximizing theproductivity of in vitro mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

Construction of Nucleic Acid Encoding Immunoadhesin

When preparing the immunoadhesins of the present invention, preferablynucleic acid encoding an extracellular domain of a natural receptor isfused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible. Typically, in such fusions the encoded chimericpolypeptide will retain at least functionally active hinge, CH2 and CH3domains of the constant region of an immunoglobulin heavy chain. Fusionsare also made to the C-terminus of the Fc portion of a constant domain,or immediately N-terminal to the CH1 of the heavy chain or thecorresponding region of the light chain. The resultant DNA fusionconstruct is expressed in appropriate host cells.

Nucleic acid molecules encoding amino acid sequence variants of nativesequence extracellular domains (such as the extracellular domain fromALK-1) and/or the antibody sequences used to prepare the desiredimmunoadhesin, are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of native sequence ALK-1.

Amino acid sequence variants of native sequence extracellular domainincluded in the chimeric heteroadhesin are prepared by introducingappropriate nucleotide changes into the native extracellular domain DNAsequence, or by in vitro synthesis of the desired chimeric heteroadhesinmonomer polypeptide. Such variants include, for example, deletions from,or insertions or substitutions of, residues in the amino acid sequenceof the immunoadhesin or chimeric heteroadhesin.

Variations in the native sequence as described above can be made usingany of the techniques and guidelines for conservative andnon-conservative mutations.

In one embodiment, the nucleic acid encodes a chimeric molecule in whichthe ALK-1 receptor extracellular domain sequence is fused to theN-terminus of the C-terminal portion of an antibody (in particular theFc domain), containing the effector functions of an immunoglobulin, e.g.IgG₁. It is possible to fuse the entire heavy chain constant region tothe ALK-1 receptor extracellular domain sequence. However, morepreferably, a sequence beginning in the hinge region just upstream ofthe papain cleavage site (which defines IgG Fc chemically; residue 216,taking the first residue of heavy chain constant region to be 114 (Kobetet al., supra), or analogous sites of other immunoglobulins) is used inthe fusion. In one embodiment, the ALK-1 receptor extracellular domainsequence is fused to the hinge region and CH2 and CH3 or CH1, hinge, CH2and CH3 domains of an IgG₁, IgG₂, or IgG₃ heavy chain. The precise siteat which the fusion is made is not critical, and the optimal site can bedetermined by routine experimentation.

For human immunoadhesins, the use of human IgG₁, and IgG₃ immunoglobulinsequences is preferred. A major advantage of using IgG₁ is that IgG₁immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG₃ requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG₃ hingeis longer and more flexible, so it can accommodate larger “adhesin”domains that may not fold or function properly when fused to IgG₁.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit.

For ALK-1 immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG₁, IgG₂ and IgG₄ all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG₄ does not activate complement,and IgG₂ is significantly weaker at complement activation than IgG₁.Moreover, unlike IgG₁, IgG₂ does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG₃ is optimal for complement activation,its in vivo half-life in approximately one third of the other IgGisotypes.

Another important consideration for immunoadhesins designed to be usedas human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG₁ hasonly four serologically-defined allotypic sites, two of which (G1m and2) are located in the Fc region; and one of these sites G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG₃, all of which are in the Fc region; only three ofthese sites (G3 m5, 11 and 21) have one allotype which isnonimmunogenic. Thus, the potential immunogenicity of an IgG₃immunoadhesin is greater than that of an IgG₁ immunoadhesin.

The cDNAs encoding the AKL-1 receptor sequence (e.g. an extracellulardomain sequence) and the Ig parts of the immunoadhesin are inserted intandem into a plasmid vector that directs efficient expression in thechosen host cells. For expression in mammalian cells pRK5-based vectors(Schall et al., Cell 61, 361-370 (1990)) and CDM8-based vectors (Seed,Nature 329, 840 (1989)) may, for example, be used. The exact junctioncan be created by removing the extra sequences between the designedjunction codons using oligonucleotide-directed deletional mutagenesis(Zoller and Smith, Nucleic Acids Res. 10, 6487 (1982); Capon et al.,Nature 337, 525-531 (1989)). Synthetic oligonucleotides can be used, inwhich each half is complementary to the sequence on either side of thedesired junction; ideally, these are 36 to 48-mers. Alternatively, PCRtechniques can be used to join the two parts of the molecule in-framewith an appropriate vector.

In one embodiment, a chimeric heteroadhesin polypeptide comprises afusion of a monomer of the chimeric heteroadhesin with a tag polypeptidewhich provides an epitope to which an anti-tag antibody can selectivelybind. Such epitope tagged forms of the chimeric heteroadhesin areuseful, as the presence thereof can be detected using a labeled antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe chimeric heteroadhesin to be readily purified by affinitypurification using the anti-tag antibody. Tag polypeptides and theirrespective antibodies are well known in the art. Examples include theflu HA tag polypeptide and its antibody 12CA5, (Field et al., Mol. Cell.Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D(gD) tag and its antibody (Paborsky et al., Protein Engineering3(6):547-553 (1990)).

Another type of covalent modification of a chimeric heteromultimercomprises linking a monomer polypeptide of the heteromultimer to one ofa variety of non-proteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. A chimeric heteromultimer also may beentrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for immunoadhesin production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Following transformation or transfection, the nucleic acid of theinvention may integrate into the host cell genome, or may exist as anextrachromosomal element. Methods of eukaryotic cell transfection andprokaryotic cell transformation are known to the ordinarily skilledartisan, for example, CaCl₂, CaPO₄, liposome-mediated andelectroporation. Depending on the host cell used, transformation isperformed using standard techniques appropriate to such cells. Thecalcium treatment employing calcium chloride, as described in Sambrooket al., supra, or electroporation is generally used for prokaryotes.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published 29 Jun. 1989. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham and vander Eb, Virology, 52:456-457 (1978) can be employed. General aspects ofmammalian cell host system transfections have been described in U.S.Pat. No. 4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyomithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. lichenifonnis (e.g., B. lichenifonnis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is a common host strain for recombinant DNAproduct fermentation. Preferably, the host cell secretes minimal amountsof proteolytic enzymes. For example, strain W3110 may be modified toeffect a genetic mutation in the genes encoding proteins endogenous tothe host, with examples of such hosts including E. coli W3110 strain1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4,which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7(ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompTrbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forimmunoadhesin-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2): 737-1742 [1983]), K fragilis (ATCC 12,424), Kbulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1998]); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]).Methylotropic yeasts are suitable herein and include, but are notlimited to, yeast capable of growth on methanol selected from the generaconsisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,Torulopsis, and Rhodotorula. A list of specific species that areexemplary of this class of yeasts may be found in C. Anthony, TheBiochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated immunoadhesin arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75);human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

In general, the choice of a mammalian host cell line for the expressionof ALK-1 immunoadhesins depends mainly on the expression vector. Anotherconsideration is the amount of protein that is required. Milligramquantities often can be produced by transient transfections. Forexample, the adenovirus EIA-transformed 293 human embryonic kidney cellline can be transfected transiently with pRK5-based vectors by amodification of the calcium phosphate method to allow efficientimmunoadhesin expression. CDM8-based vectors can be used to transfectCOS cells by the DEAE-dextran method (Aruffo et al., Cell 61, 1303-1313(1990); Zettmeissl et al., DNA Cell Biol. (US) 9, 347-353 (1990)). Iflarger amounts of protein are desired, the immunoadhesin can beexpressed after stable transfection of a host cell line. For example, apRK5-based vector can be introduced into Chinese hamster ovary (CHO)cells in the presence of an additional plasmid encoding dihydrofolatereductase (DHFR) and conferring resistance to G418. Clones resistant toG418 can be selected in culture; these clones are grown in the presenceof increasing levels of DHFR inhibitor methotrexate; clones areselected, in which the number of gene copies encoding the DHFR andimmunoadhesin sequences is co-amplified. If the immunoadhesin contains ahydrophobic leader sequence at its N-terminus, it is likely to beprocessed and secreted by the transfected cells. The expression ofimmunoadhesins with more complex structures may require uniquely suitedhost cells; for example, components such as light chain or J chain maybe provided by certain myeloma or hybridoma cell hosts (Gascoigne etal., 1987, supra; Martin et al., J. Virol. 67, 3561-3568 (1993)).

Selection and Use of a Replicable Vector

The nucleic acid encoding immunoadhesin may be inserted into areplicable vector for cloning (amplification of the DNA) or forexpression. Various vectors are publicly available. The vector may, forexample, be in the form of a plasmid, cosmid, viral particle, or phage.The appropriate nucleic acid sequence may be inserted into the vector bya variety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

The immunoadhesin may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, which maybe a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the immunoadhesin-encoding DNA that is inserted into the vector.The signal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus or BPV) are useful forcloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theimmunoadhesin-encoding nucleic acid, such as DHFR or thymidine kinase.An appropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)).

Expression and cloning vectors usually contain a promoter operablylinked to the immunoadhesin-encoding nucleic acid sequence to directmRNA synthesis. Promoters recognized by a variety of potential hostcells are well known. Promoters suitable for use with prokaryotic hostsinclude the β-lactamase and lactose promoter systems (Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)),alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776), and hybrid promoters suchas the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)). Promoters for use in bacterial systems also will contain aShine-Dalgarno sequence operably linked to the DNA encodingimmunoadhesin.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem., 255:2073 (1980)) or other glycolytic enzymees (Hess et al., J.Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

The transcription of immunoadhesin from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,retrovirus (such as avian sarcoma virus), cytomegalovirus, hepatitis-Bvirus and Simian Virus 40 (SV40); from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, or fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

Transcription of a DNA encoding the immunoadhesin by higher eukaryotesmay be increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, which act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (by 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theimmunoadhesin coding sequence, but is preferably located at a site 5′from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 3′ untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding immunoadhesin.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of immunoadhesin in recombinant vertebrate cell cultureare described in Gething et al., Nature, 293:620-625 (1981); Mantei etal., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

Purification and Characterization of Immunoadhesin

An immunoadhesin or a chimeric heteroadhesin preferably is recoveredfrom the culture medium as a secreted polypeptide, although it also maybe recovered from host cell lysates. As a first step, the particulatedebris, either host cells or lysed fragments, is removed, for example,by centrifugation or ultrafiltration; optionally, the protein may beconcentrated with a commercially available protein concentration filter,followed by separating the chimeric heteroadhesin from other impuritiesby one or more purification procedures selected from: fractionation onan immunoaffinity column; fractionation on an ion-exchange column;ammonium sulphate or ethanol precipitation; reverse phase HPLC;chromatography on silica; chromatography on heparin Sepharose;chromatography on a cation exchange resin; chromatofocusing; SDS-PAGE;and gel filtration.

A particularly advantageous method of purifying immunoadhesins isaffinity chromatography. The choice of affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human IgG₁, IgG₂, or IgG₄ heavy chains (Lindmark et al., J. Immunol.Meth. 62, 1-13 (1983)). Protein G is recommended for all mouse isotypesand for human IgG3 (Guss et al., EMBO J. 5, 15671575 (1986)). The matrixto which the affinity ligand is attached is most often agarose, butother matrices are also available. Mechanically stable matrices such ascontrolled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. The conditions for binding an immunoadhesin to the protein A orG affinity column are dictated entirely by the characteristics of the Fcdomain; that is, its species and isotype. Generally, when the properligand is chosen, efficient binding occurs directly from unconditionedculture fluid. One distinguishing feature of immunoadhesins is that, forhuman IgG, molecules, the binding capacity for protein A is somewhatdiminished relative to an antibody of the same Fc type. Boundimmunoadhesin can be efficiently eluted either at acidic pH (at or above3.0), or in a neutral pH buffer containing a mildly chaotropic salt.This affinity chromatography step can result in an immunoadhesinpreparation that is >95% pure.

Other methods known in the art can be used in place of, or in additionto, affinity chromatography on protein A or G to purify immunoadhesins.Immunoadhesins behave similarly to antibodies in thiophilic gelchromatography (Hutchens and Porath, Anal. Biochem. 159, 217-226 (1986))and immobilized metal chelate chromatography (Al-Mashikhi and Makai, J.Dairy Sci. 71, 1756-1763 (1988)). In contrast to antibodies, however,their behavior on ion exchange columns is dictated not only by theirisoelectric points, but also by a charge dipole that may exist in themolecules due to their chimeric nature.

Preparation of epitope tagged immunoadhesin facilitates purificationusing an immunoaffinity column containing antibody to the epitope toadsorb the fusion polypeptide.

In some embodiments, the ALK-1 immunoadhesins are assembled as monomers,or hetero- or homo-multimers, dimers or tetramers, essentially asillustrated in WO 91/08298. Generally, these assembled immunoglobulinswill have known unit structures. A basic four chain structural unit isthe form in which IgG, IgD, and IgE exist. A four-unit structure isrepeated in the higher molecular weight immunoglobulins; IgM generallyexists as a pentamer of basic four units held together by disulfidebonds. IgA globulin, and occasionally IgG globulin, may also exist inmultimeric form in serum. In the case of multimer, each four unit may bethe same or different.

Generally, the ALK-1 immunoadhesins of the invention will have any oneor more of the following properties: capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with the activities ofALK-1 including, for example, reduction or blocking of ALK-1 receptoractivation, reduction or blocking of ALK-1 downstream molecularsignaling, e.g., phosphorylation of Smad1, Smad5 and/or Smad8,disruption or blocking of ALK-1 ligand (e.g., BMP9 or BMP10) binding toALK-1.

Antibodies

In one embodiment the anti-ALK-1 antibodies are monoclonal. Alsoencompassed within the scope of the invention are Fab, Fab′, Fab′-SH andF(ab′)₂ fragments of the anti-ALK-1 antibodies provided herein. Theseantibody fragments can be created by traditional means, such asenzymatic digestion, or may be generated by recombinant techniques. Suchantibody fragments may be chimeric or humanized. These fragments areuseful for the diagnostic and therapeutic purposes set forth below.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The anti-ALK-1 monoclonal antibodies can be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to ALK-1 generally are raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of ALK-1 and an adjuvant. ALK-1 may be prepared using methodswell-known in the art, some of which are further described herein. Forexample, recombinant production of ALK-1 is described below. In oneembodiment, animals are immunized with a derivative of ALK-1 thatcontains the extracellular domain (ECD) of ALK-1 fused to the Fc portionof an immunoglobulin heavy chain. In a another embodiment, animals areimmunized with an ALK-1-IgG₁ fusion protein. Animals ordinarily areimmunized against immunogenic conjugates or derivatives of ALK-1 withmonophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (RibiImmunochem. Research, Inc., Hamilton, Mont.) and the solution isinjected intradermally at multiple sites. Two weeks later the animalsare boosted. 7 to 14 days later animals are bled and the serum isassayed for anti-ALK-1 titer. Animals are boosted until titer plateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against ALK-1. Preferably,the binding specificity of monoclonal antibodies produced by hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbentassay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The anti-ALK-1 antibodies can be made by using combinatorial librariesto screen for synthetic antibody clones with the desired activity oractivities. In principle, synthetic antibody clones are selected byscreening phage libraries containing phage that display variousfragments of antibody variable region (Fv) fused to phage coat protein.Such phage libraries are panned by affinity chromatography against thedesired antigen. Clones expressing Fv fragments capable of binding tothe desired antigen are adsorbed to the antigen and thus separated fromthe non-binding clones in the library. The binding clones are theneluted from the antigen, and can be further enriched by additionalcycles of antigen adsorption/elution. Any of the anti-ALK-1 antibodiescan be obtained by designing a suitable antigen screening procedure toselect for the phage clone of interest followed by construction of afull length anti-ALK-1 antibody clone using the Fv sequences from thephage clone of interest and suitable constant region (Fc) sequencesdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991),vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g. asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g. as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-ALK-1 clones is desired, the subject is immunized withALK-1 to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-ALK-1 clones isobtained by generating an anti-ALK-1 antibody response in transgenicmice carrying a functional human immunoglobulin gene array (and lackinga functional endogenous antibody production system) such that ALK-1immunization gives rise to B cells producing human antibodies againstALK-1. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-ALK-1 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing ALK-1-specific membrane bound antibody, e.g., by cellseparation with ALK-1 affinity chromatography or adsorption of cells tofluorochrome-labeled ALK-1 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which ALK-1 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al. (1989), orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVK and VX segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (Kd-1 of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (Kd-1 of about 106 to 107 M-1), but affinitymaturation can also be mimicked in vitro by constructing and reselectingfrom secondary libraries as described in Winter et al. (1994), supra.For example, mutation can be introduced at random in vitro by usingerror-prone polymerase (reported in Leung et al., Technique, 1: 11-15(1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896(1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992). Additionally, affinity maturation can be performed byrandomly mutating one or more CDRs, e.g. using PCR with primers carryingrandom sequence spanning the CDR of interest, in selected individual Fvclones and screening for higher affinity clones. WO 9607754 (published14 Mar. 1996) described a method for inducing mutagenesis in acomplementarity determining region of an immunoglobulin light chain tocreate a library of light chain genes. Another effective approach is torecombine the VH or VL domains selected by phage display withrepertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

ALK-1 nucleic acid and amino acid sequences are known in the art and arefurther discussed herein. DNAs encoding ALK-1 can be prepared by avariety of methods known in the art. These methods include, but are notlimited to, chemical synthesis by any of the methods described in Engelset al., Agnew. Chem. Int. Ed. Engl., 28: 716-734 (1989), such as thetriester, phosphite, phosphoramidite and H-phosphonate methods. In oneembodiment, codons preferred by the expression host cell are used in thedesign of the ALK-1 encoding DNA. Alternatively, DNA encoding the ALK-1can be isolated from a genomic or cDNA library.

Following construction of the DNA molecule encoding the ALK-1, the DNAmolecule is operably linked to an expression control sequence in anexpression vector, such as a plasmid, wherein the control sequence isrecognized by a host cell transformed with the vector. In general,plasmid vectors contain replication and control sequences which arederived from species compatible with the host cell. The vectorordinarily carries a replication site, as well as sequences which encodeproteins that are capable of providing phenotypic selection intransformed cells. Suitable vectors for expression in prokaryotic andeukaryotic host cells are known in the art and some are furtherdescribed herein. Eukaryotic organisms, such as yeasts, or cells derivedfrom multicellular organisms, such as mammals, may be used.

Optionally, the DNA encoding the ALK-1 is operably linked to a secretoryleader sequence resulting in secretion of the expression product by thehost cell into the culture medium. Examples of secretory leadersequences include stII, ecotin, lamB, herpes GD, lpp, alkalinephosphatase, invertase, and alpha factor. Also suitable for use hereinis the 36 amino acid leader sequence of protein A (Abrahmsen et al.,EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell. Methods fortransfection are well known in the art, and some are further describedherein.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. Methods fortransformation are well known in the art, and some are further describedherein.

Prokaryotic host cells used to produce the ALK-1 can be cultured asdescribed generally in Sambrook et al., supra.

The mammalian host cells used to produce the ALK-1 can be cultured in avariety of media, which is well known in the art and some of which isdescribed herein.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

Purification of ALK-1 may be accomplished using art-recognized methods,some of which are described herein.

The purified ALK-1 can be attached to a suitable matrix such as agarosebeads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyl methacrylate gels, polyacrylic and polymethacryliccopolymers, nylon, neutral and ionic carriers, and the like, for use inthe affinity chromatographic separation of phage display clones.Attachment of the ALK-1 protein to the matrix can be accomplished by themethods described in Methods in Enzymology, vol. 44 (1976). A commonlyemployed technique for attaching protein ligands to polysaccharidematrices, e.g. agarose, dextran or cellulose, involves activation of thecarrier with cyanogen halides and subsequent coupling of the peptideligand's primary aliphatic or aromatic amines to the activated matrix.

Alternatively, ALK-1 can be used to coat the wells of adsorption plates,expressed on host cells affixed to adsorption plates or used in cellsorting, or conjugated to biotin for capture with streptavidin-coatedbeads, or used in any other art-known method for panning phage displaylibraries.

The phage library samples are contacted with immobilized ALK-1 underconditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by ALK-1 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1.000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for ALK-1.However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting ALK-1, rare high affinity phage could becompeted out. To retain all the higher affinity mutants, phages can beincubated with excess biotinylated ALK-1, but with the biotinylatedALK-1 at a concentration of lower molarity than the target molaraffinity constant for ALK-1. The high affinity-binding phages can thenbe captured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphages with lower affinity. Conditions used in washing phages bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics.

Anti-ALK-1 clones may be activity selected. In one embodiment, theinvention provides anti-ALK-1 antibodies that reduce or block ALK-1receptor activation, reduce or block ALK-1 downstream molecularsignaling, e.g., phosphorylation of Smad1, Smad5 and/or Smad8 or disruptor block ALK-1 ligand (e.g., BMP9 or BMP10) binding to ALK-1.

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones is readily isolated and sequenced using conventionalprocedures (e.g. by using oligonucleotide primers designed tospecifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones can be combined with known DNA sequencesencoding heavy chain and/or light chain constant regions (e.g. theappropriate DNA sequences can be obtained from Kabat et al., supra) toform clones encoding full or partial length heavy and/or light chains.It will be appreciated that constant regions of any isotype can be usedfor this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In a preferredembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding anti-ALK-1 antibody derived from a hybridoma can also bemodified, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of homologous murinesequences derived from the hybridoma clone (e.g. as in the method ofMorrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNAencoding a hybridoma or Fv clone-derived antibody or fragment can befurther modified by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In this manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of the Fv clone or hybridomaclone-derived antibodies.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Human Antibodies

Human anti-ALK-1 antibodies can be constructed by combining Fv clonevariable domain sequence(s) selected from human-derived phage displaylibraries with known human constant domain sequences(s) as describedabove. Alternatively, human monoclonal anti-ALK-1 antibodies can be madeby the hybridoma method. Human myeloma and mouse-human heteromyelomacell lines for the production of human monoclonal antibodies have beendescribed, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forALK-1 and the other is for any other antigen. Exemplary bispecificantibodies may bind to two different epitopes of the ALK-1 protein.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express ALK-1. These antibodies possess an ALK-1-binding armand an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)2 bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the CH3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g. alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fe region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc,wherein VD1 is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fcregion chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

The preferred glycosylation variant herein comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 2 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 2,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg, Gln, Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: asp, glu;

(4) basic: his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides, thereby generating a Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG₁, IgG₂, IgG₃ or IgG₄ Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc reg on with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

Antibody Derivatives

The antibodies can be further modified to contain additionalnonproteinaceous moieties that are known in the art and readilyavailable. Preferably, the moieties suitable for derivatization of theantibody are water soluble polymers. Non-limiting examples of watersoluble polymers include, but are not limited to, polyethylene glycol(PEG), copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Screening for Antibodies with Desired Properties

The antibodies can be characterized for their physical/chemicalproperties and biological functions by various assays known in the art.In some embodiments, antibodies are characterized for any one or more ofbinding to ALK-1, reduction or blocking of ALK-1 receptor activation,reduction or blocking of ALK-1 receptor downstream molecular signaling(e.g., phosphorylation of Smad1, Smad5 and/or Samd8), disruption orblocking of ALK-1 ligand binding to ALK-1 (e.g., BMP9 or BMP10),inhibition of angiogenesis, inhibition of lymphangiogenesis, treatmentand/or prevention of a tumor, cell proliferative disorder or a cancer,treatment or prevention of a disorder associated with ALK-1 expressionand/or activity.

The purified antibodies can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies of the present invention are tested for their antigen bindingactivity. The antigen binding assays that are known in the art and canbe used herein include without limitation any direct or competitivebinding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays.

In one embodiment, the antibody is an altered antibody that possessessome but not all effector functions, which make it a desired candidatefor many applications in which the half life of the antibody in vivo isimportant yet certain effector functions (such as complement and ADCC)are unnecessary or deleterious. In certain embodiments, the Fcactivities of the produced immunoglobulin are measured to ensure thatonly the desired properties are maintained. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitroassay to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).C1q binding assays may also be carried out to confirm that the antibodyis unable to bind C1q and hence lacks CDC activity. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed. FcRn binding and invivo clearance/half life determinations can also be performed usingmethods known in the art, e.g. those described in the Examples section.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding the antibodyis readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Manyvectors are available. The choice of vector depends in part on the hostcell to be used. Generally, preferred host cells are of eitherprokaryotic or eukaryotic (generally mammalian) origin. It will beappreciated that constant regions of any isotype can be used for thispurpose, including IgG, IgM, IgA, IgD, and IgE constant regions, andthat such constant regions can be obtained from any human or animalspecies.

Generating Antibodies Using Prokaryotic Host Cells:

Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodycan be obtained using standard recombinant techniques. Desiredpolynucleotide sequences may be isolated and sequenced from antibodyproducing cells such as hybridoma cells. Alternatively, polynucleotidescan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in prokaryotic hosts. Many vectors that are availableand known in the art can be used for the purpose of the presentinvention. Selection of an appropriate vector will depend mainly on thesize of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector may comprise two or more promoter-cistron pairs,encoding each of the polypeptide components. A promoter is anuntranslated regulatory sequence located upstream (5′) to a cistron thatmodulates its expression. Prokaryotic promoters typically fall into twoclasses, inducible and constitutive. Inducible promoter is a promoterthat initiates increased levels of transcription of the cistron underits control in response to changes in the culture condition, e.g. thepresence or absence of a nutrient or a change in temperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector. Both the native promotersequence and many heterologous promoters may be used to directamplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing antibodies includeArchaebacteria and Eubacteria, such as Gram-negative or Gram-positiveorganisms. Examples of useful bacteria include Escherichia (e.g., E.coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Inone embodiment, gram-negative cells are used. In one embodiment, E. colicells are used as hosts for the invention. Examples of E. coli strainsinclude strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2(Washington, D.C.: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, includingstrain 33D3 having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strainsand derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B,E. coliλ 1776 (ATCC 31,537) and E. coli RV308(ATCC 31,608) are alsosuitable. These examples are illustrative rather than limiting. Methodsfor constructing derivatives of any of the above-mentioned bacteriahaving defined genotypes are known in the art and described in, forexample, Bass et al., Proteins, 8:309-314 (1990). It is generallynecessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. coli, Serratia, or Salmonella species can be suitably used as thehost when well known plasmids such as pBR322, pBR325, pACYC 177, orpKN410 are used to supply the replicon. Typically the host cell shouldsecrete minimal amounts of proteolytic enzymes, and additional proteaseinhibitors may desirably be incorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides are grown in mediaknown in the art and suitable for culture of the selected host cells.Examples of suitable media include luria broth (LB) plus necessarynutrient supplements. In some embodiments, the media also contains aselection agent, chosen based on the construction of the expressionvector, to selectively permit growth of prokaryotic cells containing theexpression vector. For example, ampicillin is added to media for growthof cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector, proteinexpression is induced under conditions suitable for the activation ofthe promoter. In one aspect of the invention, PhoA promoters are usedfor controlling transcription of the polypeptides. Accordingly, thetransformed host cells are cultured in a phosphate-limiting medium forinduction. Preferably, the phosphate-limiting medium is the C.R.A.Pmedium (see, e.g., Simmons et al., J. Immunol. Methods (2002),263:133-147). A variety of other inducers may be used, according to thevector construct employed, as is known in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides, variousfermentation conditions can be modified. For example, to improve theproper assembly and folding of the secreted antibody polypeptides,additional vectors overexpressing chaperone proteins, such as Dsbproteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolylcis,trans-isomerase with chaperone activity) can be used to co-transformthe host prokaryotic cells. The chaperone proteins have beendemonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system.

Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized is preferably a column comprising a glass or silicasurface, more preferably a controlled pore glass column or a silicicacid column. In some applications, the column has been coated with areagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Immunoconjugates

The invention contemplates immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising an anti-ALK-1 antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may affect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/ldec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and 111In or 90Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (e.g., see above). Enzymatically active toxins andfragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothio lane(IT), bifunctional derivatives of imido esters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA. 1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA. 1-maytansinoidconjugate was tested in vitro on the human breast cancer cell line SK−BR-3, which expresses 3×105 HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 15:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ1I,α2I, α3I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which is anantifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc99m or I123, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-11, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc⁹⁹m or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds expressly contemplate, but are not limited to, ADCprepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). Seepages 467-498, 2003-2004 Applications Handbook and Catalog.

Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC), an antibody (Ab) is conjugated toone or more drug moieties (D), e.g. about 1 to about 20 drug moietiesper antibody, through a linker (L). The ADC of Formula I may be preparedby several routes, employing organic chemistry reactions, conditions,and reagents known to those skilled in the art, including: (1) reactionof a nucleophilic group of an antibody with a bivalent linker reagent,to form Ab-L, via a covalent bond, followed by reaction with a drugmoiety D; and (2) reaction of a nucleophilic group of a drug moiety witha bivalent linker reagent, to form D-L, via a covalent bond, followed byreaction with the nucleophilic group of an antibody. Additional methodsfor preparing ADC are described herein.

Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither glactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Covalent Modifications to ALK-1 Polypeptides

Covalent modifications of the polypeptide antagonists of the invention(e.g., a polypeptide antagonist fragment, an ALK-1 fusion molecule, suchas an ALK-1 immunoadhesin, or an anti-ALK-1 antibody), are includedwithin the scope of this invention. They may be made by chemicalsynthesis or by enzymatic or chemical cleavage of the polypeptide, ifapplicable. Other types of covalent modifications of the polypeptide areintroduced into the molecule by reacting targeted amino acid residues ofthe polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues, orby incorporating a modified amino acid or unnatural amino acid into thegrowing polypeptide chain, e.g., Ellman et al. Meth. Enzyme 202:301-336(1991); Noren et al. Science 244:182 (1989); and, & US Patentapplication publications 20030108885 and 20030082575.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction istypically performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T.E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to a polypeptide of the invention.These procedures are advantageous in that they do not require productionof the polypeptide in a host cell that has glycosylation capabilitiesfor N- or O-linked glycosylation. Depending on the coupling mode used,the sugar(s) may be attached to (a) arginine and histidine, (b) freecarboxyl groups, (c) free sulfhydryl groups such as those of cysteine,(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 published 11 Sep. 1987, and inAplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on a polypeptide of theinvention may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddin,et al. Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al. Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties,e.g., on antibodies, can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al. Meth. Enzymol.138:350 (1987).

Another type of covalent modification of a polypeptide of the inventioncomprises linking the polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody or immunoadhesin of theinvention are prepared for storage by mixing the antibody having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington: The Science and Practiceof Pharmacy 20th edition (2000)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,histidine and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin, which matrices arein the form of shaped articles, e.g., films, or microcapsule. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulatedimmunoglobulins remain in the body for a long time, they may denature oraggregate as a result of exposure to moisture at 37° C., resulting in aloss of biological activity and possible changes in immunogenicity.Rational strategies can be devised for stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

It is further contemplated that an agent useful in the invention can beintroduced to a subject by gene therapy. Gene therapy refers to therapyperformed by the administration of a nucleic acid to a subject. In genetherapy applications, genes are introduced into cells in order toachieve in vivo synthesis of a therapeutically effective geneticproduct, for example for replacement of a defective gene. “Gene therapy”includes both conventional gene therapy where a lasting effect isachieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs or siRNA can be used as therapeutic agents for blockingthe expression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 (1986)). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups. For general reviewsof the methods of gene therapy, see, for example, Goldspiel et al.Clinical Pharmacy 12:488-505 (1993); Wu and Wu Biotherapy 3:87-95(1991); Tolstoshev Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan Science 260:926-932 (1993); Morgan and Anderson Ann. Rev.Biochem. 62:191-217 (1993); and May TIBTECH 11:155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. eds. (1993) Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler (1990) GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

Dosage and Administration

The molecules are administered to a human patient, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes, and/or subcutaneousadministration.

In certain embodiments, the treatment of the invention involves thecombined administration of an ALK-1 antagonist and one or moreanti-cancer agents, e.g., anti-angiogenesis agents oranti-lymphangiogenesis agents. In one embodiment, additional anti-canceragents are present, e.g., one or more different anti-angiogenesisagents, one or more chemotherapeutic agents, etc. The invention alsocontemplates administration of multiple inhibitors, e.g., multipleantibodies to the same antigen or multiple antibodies to differentcancer active molecules. In one embodiment, a cocktail of differentchemotherapeutic agents is administered with the ALK-1 antagonist and/orone or more anti-angiogenesis agents. The combined administrationincludes coadministration, using separate formulations or a singlepharmaceutical formulation, and/or consecutive administration in eitherorder. For example, a ALK-1 antagonist may precede, follow, alternatewith administration of the anti-cancer agents, or may be givensimultaneously therewith. In one embodiment, there is a time periodwhile both (or all) active agents simultaneously exert their biologicalactivities.

For the prevention or treatment of disease, the appropriate dosage ofALK-1 antagonist will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theinhibitor is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theinhibitor, and the discretion of the attending physician. The inhibitoris suitably administered to the patient at one time or over a series oftreatments. In a combination therapy regimen, the compositions of theinvention are administered in a therapeutically effective amount or atherapeutically synergistic amount. As used herein, a therapeuticallyeffective amount is such that administration of a composition of theinvention and/or co-administration of ALK-1 antagonist and one or moreother therapeutic agents, results in reduction or inhibition of thetargeting disease or condition. The effect of the administration of acombination of agents can be additive. In one embodiment, the result ofthe administration is a synergistic effect. A therapeuticallysynergistic amount is that amount of ALK-1 antagonist and one or moreother therapeutic agents, e.g., an angiogenesis inhibitor, necessary tosynergistically or significantly reduce or eliminate conditions orsymptoms associated with a particular disease.

Depending on the type and severity of the disease, about 1 μg/kg to 50mg/kg (e.g. 0.1-20 mg/kg) of ALK-1 antagonist or angiogenesis inhibitoris an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. A typical daily dosage might range from about 1μg/kg to about 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. Typically, the clinician will administered a molecule(s)until a dosage(s) is reached that provides the required biologicaleffect. The progress of the therapy of the invention is easily monitoredby conventional techniques and assays.

For example, preparation and dosing schedules for angiogenesisinhibitors, e.g., anti-VEGF antibodies, such as AVASTIN® (Genentech),may be used according to manufacturers' instructions or determinedempirically by the skilled practitioner. Depending on the type andseverity of the disease, about 1 μg/kg to 100 mg/kg (e.g., 0.1-20 mg/kg)of VEGF-specific antagonist is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. In some embodiments,particularly desirable dosages include, for example, 5 mg/kg, 7.5 mg/kg,10 mg/kg, and 15 mg/kg. For repeated administrations over several daysor longer, depending on the condition, the treatment is sustained untilthe cancer is treated, as measured by the methods described above orknown in the art. However, other dosage regimens may be useful. In oneexample, if the VEGF-specific antagonist is an antibody, the antibody ofthe invention is administered once every week, every two weeks, or everythree weeks, at a dose range from about 5 mg/kg to about 15 mg/kg,including but not limited to 7.5 mg/kg or 10 mg/kg. The progress of thetherapy of the invention is easily monitored by conventional techniquesand assays.

In one example, bevacizumab is the VEGF-specific antagonist. Bevacizumabis supplied for therapeutic uses in 100 mg and 400 mg preservative-free,single-use vials to deliver 4 ml or 16 ml of bevacizumab (25 mg/ml). The100 mg product is formulated in 240 mg α,α-trehalose dehydrate, 23.2 mgsodium phosphate (monobasic, monohydrate), 4.8 mg sodium phosphate(dibasic, anhydrous), 1.6 mg polysorbate 20, and Water for Injection,USP. The 400 mg product is formulated in 960 mg α,α-trehalose dehydrate,92.8 mg sodium phosphate (monobasic, monohydrate), 19.2 mg sodiumphosphate (dibasic, anhydrous), 6.4 mg polysorbate 20, and Water forInjection, USP.

In another example, preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992).

Efficacy of the Treatment

The efficacy of the treatment of the invention can be measured byvarious endpoints commonly used in evaluating neoplastic ornon-neoplastic disorders. For example, cancer treatments can beevaluated by, e.g., but not limited to, tumor regression, tumor weightor size shrinkage, time to progression, duration of survival,progression free survival, overall response rate, duration of response,and quality of life. Because the anti-angiogenesis agents describedherein target the tumor vasculature and not necessarily the neoplasticcells themselves, they represent a unique class of anticancer drugs, andtherefore can require unique measures and definitions of clinicalresponses to drugs. For example, tumor shrinkage of greater than 50% ina 2-dimensional analysis is the standard cut-off for declaring aresponse. However, the inhibitors may cause inhibition of metastaticspread without shrinkage of the primary tumor, or may simply exert atumouristatic effect. Accordingly, approaches to determining efficacy ofthe therapy can be employed, including for example, measurement ofplasma or urinary markers of angiogenesis and measurement of responsethrough radiological imaging.

The following examples are provided for illustrative purposes only andare not to be construed as limiting upon the teachings herein.

EXAMPLES Example 1 Generation of an ALK1.Fc Molecule

Using standard molecular biology techniques, an ALK1.Fc molecule wasgenerated by attaching the extracellular domain of ALK-1 (amino acidresidues 1-118 of human ALK-1) to the Fc region of human IgG₁ via apolypeptide linker. Briefly, an extracellular fragment of human ALK-1(amino acid 1-118) was subcloned into a pRK5 vector that had beenengineered for the expression of fusion protein with a C-terminal Fc ofhuman IgG1. ALK1.Fc was purified with Protein-A affinity chromatographyfrom conditioned medium harvested from serum-free culture of CHO cellstransiently transfected with the expression plasmid. The ALK1.Fcmolecule had the following cDNA and amino acid sequences:

ALK1.Fc cDNA sequence: (SEQ ID NO: 1)ATGACCTTGGGCTCCCCCAGGAAAGGCCTTCTGATGCTGCTGATGGCCTTGGTGACCCAGGGAGACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAGACCGGTGTCACCGACAAAGCTGCGCACTATACTCTGTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGCCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAATGA ALK1.Fcprotein sequence: (SEQ ID NO: 2)MTLGSPRKGLLMLLMALVTQGDPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQTGVTDKAAHYTLCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK

Example 2 Treatment with ALK1.Fc Enhances Endothelial Cell Sprouting InVitro

The effect of inhibiting ALK-1 signalling on endothelial cell growth wasexplored using the HUVEC fibrin gel bead assay. Details of the HUVECfibrin gel bead assay have been previously described (Nakatsu, Minn.,Microvascular Research 44:102-112 (2003)). Briefly, Cytodex 3 beads(Amersham Pharmacia Biotech) were coated with 350-400 human umbilicalvein endothelia cells (HUVECs) per bead in 2 ml EGM-2 medium(Clonetics). About 200 LUVECs-coated beads were imbedded in fibrin clotin one well of 12-well tissue culture plate. 80×10³ D551 humanfibroblast cells were plated on top of the clot and medium was changedevery 2 days. ALK1.Fc was added to the culture medium at 5 ug/ml.Phase-contrast microscopy images were taken on day 9.

HUVECs growing in fibrin gels in the presence of co-cultured humanfibroblast cells generate sprouts with a distinct lumen-like structure(Nakatsu, M. N. et al. Microvasc Res 66, 102-12 (2003)). Addition ofALK1.Fc greatly increased the length of the sprouts (FIG. 1), suggestingthat blocking ALK-1 signaling allowed the endothelial cells to adapt amore exploratory behavior. These results indicated that ALK1.Fc was ableto affect endothelial cells by sequestering ALK-1 ligand and preventingthe activation of endogenous ALK-1.

Example 3 Treatment with ALK1-Fc Affects Retinal Vascular Development InVivo

Neonatal SD rats from the same litters were used to study the effect ofALK1.Fc on retinal vascular development. Rats were injected i.p. withALK1.Fc (10 mg/kg body weight) on postnatal day 4 (P4), P6 and P9. OnP11, rats were anesthetized with Isoflurane. FITC-labeled LycopersiconEsculentum Lectin (150 μg in 150 μl of 0.9% NaCl; Vector Laboratories)was injected intracardially and allowed to circulate for 3 min. Eyeswere collected and fixed with 4% PFA in PBS overnight, followed by PBSwashes. The dissected retinas were blocked with 10% goat serum in PBST(PBS, 0.5% Triton X-100) for 3 hrs, then incubated overnight withbiotinylated isolectin B4 (1:100, Bandeiraea simplicifolia; MolecularProbe) or Cy3-conjugated anti-alpha smooth muscle actin (ASMA, 1:200,Sigma-Aldrich), with 10% goat serum in PBST. To visualize thebiotynylated isolectin B4, retinas were then washed 4 times in PBST, andincubated overnight with Cy3-streptavidin (1:200, Sigma-Aldrich). Afterstaining was completed, retinas were washed 4 times in PBST. Allovernight incubations were done at 4° C. Images of flat mounted retinaswere captured by confocal fluorescence microscopy.

Early postnatal rat retina develops a stereotypic vascular pattern in awell-defined sequence of events. The superficial retinal vasculaturedevelops as an expanding network from the optic nerve head, with activesprouting at the periphery and extensive remodeling at the center.Different aspects of vessel development, e.g. vessel sprouting,remodeling, maturation and arterial-venous specification can be readilyfollowed. In ALK1.Fc treated retinas, the characteristic pattern ofradially alternating arteries and veins was mostly unaffected. Therewas, however, a clear increase of the vascular density, especially themicrovessels near the veins (Isolectin-Cy3; FIG. 2). In addition therewas a dramatic reduction of blood perfusion in the microvessels andveins (Lectin-FITC; FIGS. 3 & 4). Interestingly, the retinal arterieswere apparently enlarged, which was reminiscent of the vascular defectobserved in Alk1 deficient embryos. Furthermore, the enlarged retinalarteries have disorganized vascular smoother muscle cell coverage (ASMA;FIGS. 3 & 4). These results indicate that ALK1.Fc treatment was able toaffect vascular development in neonatal rat retina by interfering withALK-1 signaling in vivo.

Example 4 Treatment with ALK1-Fc Inhibits Tumor Growth In Vivo

To explore whether ALK-1 signaling is directly involved in tumor growth,we tested the effect of ALK1.Fc on tumor angiogenesis and tumor growthin nude mice bearing subcutaneous human tumor xenografts. Beige nudemice (Charles River Laboratories, Hollister, Calif.) were maintained inaccordance with the guide for the care and use of laboratory animals.Six- to eight-week-old female mice were used in each study. To obtainsubcutaneous tumors, mice were injected with 0.1 ml tumor cellsuspension containing 50% matrigel (BD Bioscience) into the rightposterior flank. 5×10⁶ human colon cancer HM7 cells, 10×10⁶ human lungcarcinoma MV-522 cells, 1×10⁶ rat glioblastoma C6 cells or 10×10⁶ humanlung carcinoma Calu6 cells were injected into each mouse. ALK1.Fc (10mg/kg) was administered via i.p., as follows: for HM7 ALK1.Fc treatment(q/2d) was initiated 3 days after tumor inoculation, for MV-522 ALK1.Fctreatment (q/2d) was initiated 16 days after tumor inoculation, for C6ALK1.Fc treatment (2×/w) was initiated 3 days after tumor inoculation,for Calu6 ALK1.Fc treatment (q/2d) was initated 22 days after tumorinoculation. Tumor growth was quantitated by caliper measurements. Tumorvolume (mm³) was determined by measuring the length (l) and width (w)and calculating the volume (V=lw²/2). 10 to 15 animals were included ineach group.

ALK1.Fc treatment significantly inhibited tumor growth as compared tovehicle treatment (FIGS. 5 & 6). The effect on tumor growth was observedin all the xenograft tumor models tested indicating that the observedanti-tumor effects were not tumor type specific.

Example 5 Combination of ALK1-Fc and Anti-VEGF

In the HM7 tumor study described above in Example 4, ALK1.Fc treatmentbegan three day after the tumor cells were injected. The tumor growthinhibition activity of ALK1.Fc was reduced when treatment (10 mg/kg atq/3d) was initiated nine days after tumor cell inoculation (FIG. 7). Inthis model treatment with anti-VEGF antibody (B20-4.1, 5 mg/kg at q/3d)was also less effective when treatment was delayed. However, significantadditive effect was observed when both agents were used in combination(FIG. 7). The combined effect of anti-VEGF antibody and ALK1.Fc was alsostudied in the LL2 xenograft model. 5×10⁶ mouse lewis lung carcinoma LL2cells were injected into each mouse and treatment with ALK1.Fc (10 mg/kgat q/2d) and anti-VEGF antibody (5 mg/kg at q/2d) was initiated 2 daysafter tumor inoculation. The results show that combination therapy inthe LL2 tumor model resulted in much greater anti-tumor efficacy incomparison to mono-therapy (FIG. 8).

The effect of anti-VEGF antibody and ALK1.Fc on tumor vascular density,as assessed by CD31 staining, was examined in the LL2 tumor model. Onday 18 after tumor cell inoculation, tumors were removed and fixed byimmersion in 1% paraformaldehyde (PFA) in PBS for 2 hr, followed byincubation in 30% sucrose overnight for cryoprotection. Samples wereembedded in OCT and sections of 40 μm thickness were prepared andstained with anti-mouse CD31 (1:50, BD Pharmingen), followed byFITC-conjugated goated anti-rat IgG. As expected, treatment withanti-VEGF antibody alone significantly reduced vascular density intreated tumors. Treatment with ALK1.Fc, however, had a marginal effecton tumor vascular density. Strikingly, treatment with both agentsresulted in an even more dramatic reduction of tumor vessel density(FIG. 9), consistent with the additive anti-tumor effect. Together,these results demonstrated that blocking of Alk1 signaling with ALK1.Fccould have broad anti-tumor efficacy, especially when combined withanti-VEGF.

Example 6 Treatment with ALK1-Fc Affects Lymphatic VasculatureDevelopment In Vivo

Neonatal CD1 mice from the same litters were used to study the effect ofALK1.Fc on lymphangiogenesis. Mice were injected i.p. with ALK1.Fc ((10mg/kg body weight) on postnatal day 1 (P1), P3 and P6, and tissue washarvested on P9. Small intestines were removed en bloc and rinsed withPBS. For tail skin, the dermis was separated from the epidermis, washedwith PBS and used in subsequent staining. Tissues were fixed for 2 hrswith 4% PFA in PBS at room temperature. After blocking with PHT1 (5%goat serum, 0.2% BSA, 0.5% Triton X-100, NaN₃ in PBS) for 2 hrs at roomtemperature, tissues were incubated overnight with rat anti-CD31 (1:50,BD Pharmingen) and rabbit polyclonal anti-LYVE-1 (1:500, Upstate), inPHT1. Secondary AlexaFluor 488-conjugated goat anti-rat (1:400,Molecular Probes) and AlexaFluor594-conjugated goat anti-rabbit (1:400,Molecular Probes) in PHT2 (2% BSA, 0.5% Triton X-100, in PBS) were usedto visualize antigen-antibody complexes. After staining was completed,tissues were washed 4 times in PBST (PBS, 0.5% Triton X-100), post fixedwith 4% PFA in PBS for 5-10 min at room temperature, followed by another4 washes in PBS. All overnight incubations were carried out at 4° C.Images of flat mounted tissues were captured by confocal fluorescencemicroscopy.

In the small intestines of ALK1.Fc treated neonatal mice, the capillaryblood vessels (CD31 staining) in the villi appeared to be normalalthough the villi were moderately shorter than the control (FIGS. 10 &11). In contrast, the lymphatic capillaries (LYVE-1 staining) wereseverely affected. They failed to extend fully into the villi and weremostly stalled at the base of villi.

The lymphatic vascular network within the neonatal mouse tail skin wasalso dramatically altered after ALK1.Fc treatment (FIG. 12). Thecharacteristic honeycomb-like pattern was mostly disrupted, with onlyfragmented and disorganized lymphatic vessels. These results indicatethat, in addition to its critical role in regulating blood vesselformation, Alk1 signaling is also essential for postnatal development oflymphatic vasculature.

Example 7 Treatment with ALK1-Fc Affects Pericyte Organization

The effect of treatment with ALK1.Fc on pericyte organization in tumorvessels was examined. Beige nude mice were injected with 0.1 ml tumorcell suspension containing 50% matrigel (BD Bioscience) into the rightposterior flank. 5×10⁶ mouse monocytic leukemia TIB68 cells(BALB/c-derived and thought to represent an early stage inmonocyte-myelocyte differentiation) were injected into each mouse toestablish subcutaneous tumors. Alk1.Fc (10 mg/kg) or control vehicle(PBS) was administered via i.p. every three days. Treatments wereinitiated the next day after cell inoculation and ended 16 days afterthe first dose. Tumors were fixed for 2 hours in 4% paraformaldehyde(PFA) at room temperature (RT), rinsed in PBS, transferred to 30%sucrose at 4° C. overnight, and embedded in OCT. 80 μm sections of thetumor tissue were washed in PBS, blocked with PHT 1 (PBS/10% goatserum/0.5% Triton X-100) at RT for 2 hours, and incubated with primaryantibodies diluted in PHT2 (PBS/2% goat serum/0.5% Triton X-100) at 4°C. overnight (rat anti-mouse CD31, 1:50, BD Pharmingen; rabbitanti-Desmin, 1:400, GeneTex). After four washes in PBS/0.2% TritonX-100, the sections were incubated with secondary antibodies (goat-antirat Alexa 488; goat anti-rabbit Alexa 594, 1:400, Molecular Probes) atRT for 2 h. Sections were washed in PBS/0.2% Triton X-100 followed byPBS, fixed with 4% PFA at RT for 5 min and finally washed with PBS.These results show that ALK1.Fc treated tumor vessels have poorlyorganized and detached pericytes (FIG. 13).

Example 8 Generation of Additional ALK1 Immunoadhesin Molecules

Standard molecular biology techniques were used to generate thefollowing additional molecules:

ALK1.Fc.2 cDNA sequence: (SEQ ID NO: 3)ATGACCTTGGGCTCCCCCAGGAAAGGCCTTCTGATGCTGCTGATGGCCTTGGTGACCCAGGGAGACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAGACCGGTGTCACCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA G ALK1.Fc.2 proteinsequence: (SEQ ID NO: 4)MTLGSPRKGLLMLLMALVTQGDPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQTGVTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ALK1.Fc.3 cDNAsequence: (SEQ ID NO: 5)ATGACCTTGGGCTCCCCCAGGAAAGGCCTTCTGATGCTGCTGATGGCCTTGGTGACCCAGGGAGACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAGACCGGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGCCGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA ALK1.Fc.3 protein sequence:(SEQ ID NO: 6) MTLGSPRKGLLMLLMALVTQGDPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ALK1.Fc.4 cDNA sequence:(SEQ ID NO: 7) ATGACCTTGGGCTCCCCCAGGAAAGGCCTTCTGATGCTGCTGATGGCCTTGGTGACCCAGGGAGACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TGA ALK1.Fc.4 proteinsequence: (SEQ ID NO: 8)MTLGSPRKGLLMLLMALVTQGDPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ALK1.Fc.5 cDNAsequence: (SEQ ID NO: 9)ATGACCTTGGGCTCCCCCAGGAAAGGCCTTCTGATGCTGCTGATGGCCTTGGTGACCCAGGGAGACCCTGTGAAGCCGTCTCGGGGCCCGCTGGTGACCTGCACGTGTGAGAGCCCACATTGCAAGGGGCCTACCTGCCGGGGGGCCTGGTGCACAGTAGTGCTGGTGCGGGAGGAGGGGAGGCACCCCCAGGAACATCGGGGCTGCGGGAACTTGCACAGGGAGCTCTGCAGGGGGCGCCCCACCGAGTTCGTCAACCACTACTGCTGCGACAGCCACCTCTGCAACCACAACGTGTCCCTGGTGCTGGAGGCCACCCAACCTCCTTCGGAGCAGCCGGGAACAGATGGCCAGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TGAG ALK1.Fc.5protein sequence: (SEQ ID NO: 10)MTLGSPRKGLLMLLMALVTQGDPVKPSRGPLVTCTCESPHCKGPTCRGAWCTVVLVREEGRHPQEHRGCGNLHRELCRGRPTEFVNHYCCDSHLCNHNVSLVLEATQPPSEQPGTDGQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 9 Treatment with ALK1 Immunoadhesins Affects Retinal VascularDevelopment In Vivo

Mice were injected i.p. with ALK1.Fc, ALK1.Fc.2, ALK1.Fc.4 or ALK1.Fc.5(10 mg/kg body weight) on postnatal day 4 (P4) and P6. On P8, mice wereanesthetized. Eyes were collected and fixed with 4% PFA in PBSovernight, followed by PBS washes. The dissected retinas were blockedwith 10% goat serum in PBST (PBS, 0.5% Triton X-100) for 3 hrs, thenincubated overnight with rat anti-CD31 (1:50, BD Pharmingen) in PBST. Tovisualize the anti-CD31, retinas were incubated with AlexaFluor488-conjugated goat anti-rat (1:400, Molecular Probes) in PBST. Afterstaining was completed, retinas were washed 4 times in PBST. Allovernight incubations were done at 4° C. Images of flat mounted retinaswere captured by confocal fluorescence microscopy.

Early postnatal mouse retina develops a stereotypic vascular pattern ina well-defined sequence of events. The superficial retinal vasculaturedevelops as an expanding network from the optic nerve head, with activesprouting at the periphery and extensive remodeling at the center.Different aspects of vessel development, e.g. vessel sprouting,remodeling, maturation and arterial-venous specification can be readilyfollowed. In ALK1 immunoadhesin (ALK1.Fc, ALK1.Fc.2, ALK1.Fc.4 andALK1.Fc.5) treated retinas, the characteristic pattern of radiallyalternating arteries and veins was mostly unaffected. There was,however, a clear increase of the vascular density, especially themicrovessels near the veins (FIG. 14). These results indicate that theeffects of treatment with the additional ALK1 immunoadhesins (ALK1.Fc.2,ALK1.Fc.4 and ALK1.Fc.5) on vascular development in neonatal mouseretina are similar to treatment with ALK1.Fc.

1. A method of inhibiting lymphangiogenesis comprising administering toa subject in need of inhibition of lymphangiogenesis an effective amountof an ALK-1 antagonist, whereby the lymphangiogensis is inhibited. 2.The method of claim 1, wherein the subject suffers from a tumor, cancer,cell proliferative disorder, macular degeneration, inflammatory mediateddisease, rheumatoid arthritis, diabetic retinopathy or psoriasis.
 3. Amethod for treating a pathological condition associated withlymphangiogenesis in a subject comprising administering to the subjectan effective amount of an ALK-1 antagonist, whereby the pathologicalcondition associated with lymphangiogenesis is treated.
 4. The method ofclaim 3, wherein the pathological condition associated withlymphangiogenesis is a tumor, cancer, cell proliferative disorder,macular degeneration, inflammatory mediated disease, rheumatoidarthritis, diabetic retinopathy or psoriasis.
 5. The method of claim 2or 4, wherein the tumor, cancer or cell proliferative disorder iscarcinoma, lymphoma, blastoma, sarcoma, or leukemia.
 6. A method ofinhibiting tumoral lymphangiogenesis in a subject comprisingadministering to the subject an effective amount of an ALK-1 antagonist,whereby the tumoral lymphangiogensis is inhibited.
 7. A method ofinhibiting or preventing tumor metastasis in a subject comprisingadministering to the subject an effective amount of an ALK-1 antagonist,whereby the tumor metastasis is inhibited or prevented.
 8. The method ofclaim 6 or 7, wherein the subject has developed or is at risk fordeveloping tumor metastasis.
 9. The method of claim 8, wherein saidtumor metastasis is in the lymphatic system.
 10. The method of claim 8,wherein said tumor metastasis is in a distant organ.
 11. A method ofdisrupting pericyte organization in a subject comprising administeringto the subject an effective amount of an ALK-1 antagonist, whereby thepericyte organization is disrupted.
 12. The method of claim 11, whereinthe subject suffers from a tumor, cancer, cell proliferative disorder,macular degeneration, inflammatory mediated disease, rheumatoidarthritis, diabetic retinopathy or psoriasis.
 13. A method of inhibitingtumor growth in a subject comprising administering to the subject aneffective amount of an ALK-1 antagonist, whereby the tumor growth isinhibited.
 14. A method of treating a tumor, cancer or cellproliferative disorder in a subject comprising administering to thesubject an effective amount of an ALK-1 antagonist, whereby the tumor,cancer or cell proliferative disorder is treated.
 15. The method ofclaim 13 or 14, wherein the tumor, cancer or cell proliferative disorderis carcinoma, lymphoma, blastoma, sarcoma, or leukemia.
 16. The methodof claim 13 or 14, further comprising administering to the subject aneffective amount of an anti-angiogenesis agent.
 17. The method of claim16, wherein the anti-angiogenesis agent is an antagonist of vascularendothelial growth factor (VEGF).
 18. The method of claim 17, whereinthe antagonist of VEGF is an anti-VEGF antibody.
 19. The method of claim18, wherein the anti-VEGF antibody is bevacizumab.
 20. A method ofenhancing efficacy of an anti-angiogenesis agent in a subject having apathological condition associated with angiogenesis, comprisingadministering to the subject an effective amount of an ALK-1 antagonistin combination with the anti-angiogenesis agent, thereby enhancing saidanti-angiogenesis agent's inhibitory activity.
 21. The method of claim20, wherein the pathological condition associated with angiogenesis is atumor, cancer or cell proliferative disorder.
 22. The method of any oneof claims 1-21, wherein the ALK-1 antagonist is an ALK-1 immunoadhesin.23. The method of claim 22, wherein the ALK-1 immunoadhesin comprisesamino acid residues 22-352 of SEQ ID NO: 2, residues 22-349 of SEQ IDNO: 4, residues 22-347 of SEQ ID NO: 6, residues 22-350 of SEQ ID NO: 8or residues 22-350 of SEQ ID NO:
 10. 24. The method of any one of claims1-21, wherein the ALK-1 antagonist is an anti-ALK-1 antibody orantigen-binding fragment thereof.
 25. An ALK-1 antagonist for use in theprevention, inhibition or treatment of tumor metastasis or treatment oftumor, cancer or cell proliferative disorder.
 26. The ALK-1 antagonistof claim 25, wherein the ALK-1 antagonist is an ALK-1 immunoadhesin. 27.The ALK-1 antagonist of claim 26, wherein the ALK-1 immunoadhesincomprises amino acid residues 22-352 of SEQ ID NO: 2, resiudes 22-349 ofSEQ ID NO: 4, residues 22-347 of SEQ ID NO: 6, residues 22-350 of SEQ IDNO: 8 or residues 22-350 of SEQ ID NO:
 10. 28. The ALK-1 antagonist ofclaim 25, wherein the ALK-1 antagonist is an anti-ALK-1 antibody orantigen-binding fragment thereof.